P. aeruginosa is a multidrug resistant opportunistic pathogen which causes acute or chronic infection in immunocompromised individuals, cystic fibrosis, burns and sepsis. P. aeruginosa is able to form biofilm, which helps bacteria survive in a hypoxic atmosphere or other extremely harsh environments. Treatment of P. aeruginosa infection is extremely difficult due to its rapid mutations and adaptation to gain resistance to antibiotics and consequently, it has been listed among the critical group of pathogens which urgently need new strategies to control. One of these strategies that has been recently employed is phage therapy using bacteriophages to limit bacterial infections. Phage therapy has received a significant interest as an alternative to antibiotics and to alleviate antimicrobial resistance. Bacteriophages are seemed to be efficient alternatives in management of infections caused by P. aeruginosa. The current study aimed to isolate and characterize new lytic phages targeting antibiotic resistant P. aeruginosa that would be helpful to combat these infections that threaten human healthcare. A total of 50 P. aeruginosa isolates in this study were recovered from 120 clinical specimens during the period from April 2021 to August 2021 from patients admitted to Zagazig University Hospitals and El-Ahrar Educational Hospital, Zagazig, El Sharkia, Egypt. P. aeruginosa were isolated from different clinical sources including burns, surgical wounds, urine, ear infections and endotracheal aspirates. Antibiotic susceptibility testing was done by the standard disc diffusion method. All P. aeruginosa isolates were sensitive to colistin while higher bacterial resistance was observed towards gentamicin, fluoroquinolone and carbapenems (60, 58 and 54%; respectively). Intermediate bacterial resistance was observed against pipracillin (46%), ceftazidime (40%) and piperacillin/tazobactam (38%). Conversely, low bacterial resistance was recorded against aztreonam (18%). It is worth mentioning that 56% of P. aeruginosa isolates in this study were MDR. The biofilm forming capacity of P. aeruginosa clinical isolates in addition to P. aeruginosa reference strains; P. aeruginosa ATCC 27853, P. aeruginosa ATCC 9027 & P. aeruginosa PAO1 were assessed spectrophotometrically by the crystal violet assay. Almost all P. aeruginosa isolates were found to be capable of forming biofilm with variable degrees; 20.4% were strong biofilm producers, 64.8% were moderate biofilm producers and 14.8% were weak biofilm producers. Five lytic phages targeting P. aeruginosa isolates were isolated and fully characterized from sewage. These phages were designated as vB_PaeP_PS28 & vB_PaeP_PS49 & vB_PaeP_PS14 & vB_PaeM_PS3 and vB_PaeM_PS38. Phage particles morphology was characterized using the transmission electron microscope (TEM). TEM images revealed that the phage vB_PaeP_PS28 & vB_PaeP_PS49 and vB_PaeP_PS14 possess an icosahedral head and short non-contractile tail which are closely related to phages belonging to the family Podoviridae and order Caudovirales according to International Committee Taxonomy of Viruses (ICTV). Meanwhile, vB_PaeM_PS3 and vB_PaeM_PS38 possess an icosahedral head and contractile tails and therefore were found to belong to the family Myoviridae and the order Caudovirales. Physical stability of isolated phages was determined against wide range of pH and temperature degrees. Isolated phages were tolerant to wide range of temperatures. However, isolated phages; vB_PaeP_PS28 and vB_PaeM_PS3 could survive up to 60°C and lost their infectivity at higher temperatures (70, 80, 90 and 100°C). On the other hand, vB_PaeP_PS49 & vB_PaeP_PS14 and vB_PaeM_PS38 were considerably stable at temperatures range from 40°C to 70°C. However, their activity was entirely diminished when incubated at 80-100°C. Furthermore, the impact of different pH on survival and infectivity of all isolated phages was determined. It was found that all phages were able to retain their infectivity and could survive at wide pH ranges (4-10). There was a slight reduction in phage viability at pH 11. Of note that, no viable phage particles were observed at extreme pH (3 and 12) which means that the phages completely lost their infectivity. The host range of vB_PaeP_PS28 & vB_PaeP_PS49 & vB_PaeP_PS14 & vB_PaeM_PS3 and vB_PaeM_PS38 phages was determined against a total of 18 P. aeruginosa strains using the spot assay. Moreover, other bacterial species such as E. coli, S. Typhimurium, K. pneumoniae, Serratia marcescens and S. aureus were included in host range determination. Results show that isolated phages have a unique lytic profile and were able to infect most of tested P. aeruginosa strains indicating a higher lytic efficiency of isolated phages. However, no lytic activity was observed against other bacterial species indicating that the isolated phages possessed broad spectrum lytic activity and high specificity towards P. aeruginosa. The antibiofilm activity of isolated phages at different MOIs (0.1, 1 and 10) P. aeruginosa was evaluated by the crystal violet assay. The isolated phages effectively degraded mature biofilms and reduced biofilm biomass formed by all tested P. aeruginosa strains. The antibiofilm activity of isolated phages against P. aeruginosa was MOI-dependent where maximum biofilm inhibition was observed at MOI of 10 as compared with MOI of 1 and 0.1. Moreover, the efficiency of plating (EOP) of two phages; vB_PaeP_PS28 and vB_PaeM_PS3 was performed for each susceptible strain that showed lysis in the spot assay. Both phages were able to infect all susceptible P. aeruginosa strains with different infectivity patterns. Furthermore, one-step growth curve was performed to characterize the phage infection process including the latent period and burst size. The phage vB_PaeP_PS28 has a latent period of 15 min and an average burst size of 210 virions per infected bacterium. On the other hand, vB_PaeM_PS3 exhibited a short latent period of 10 min with host-cell lysis releasing about 132 new virions per infected cell. Furthermore, the bacteriolytic activity of both vB_PaeP_PS28 and vB_PaeM_PS3 was determined against P. aeruginosa strains at different MOIs (0.1, 1 and 10) and bacterial turbidity was measured at OD600 over 24 hr. Both vB_PaeP_PS28 and vB_PaeM_PS3 were found to adversely affect bacterial growth over 24 hr. Of note that, bacterial growth inhibition was found to be dose-dependent where growth inhibition was higher at MOI of 10 as compared to MOI of 1 and 0.1. Importantly, the whole genome of both vB_PaeP_PS28 and vB_PaeM_PS3 were sequenced by Illumina Miseq next-generation sequencing. In brief, the genome of vB_PaeP_PS28 consists of 72,283 bp circular double-stranded DNA, with G+C content of 54.75 %. The phage genome harbors 94 open reading frames (ORFs); 32 for known functional proteins and 62 for hypothetical proteins and no tRNA genes. Meanwhile the genome of vB_PaeM_PS3 consists of 93,922 bp of dsDNA with 49.39% G+C content. A total of 171 ORFs were identified, of which 27 were assigned as functional proteins whereas hypothetical proteins were encoded by 144 ORFs and 14 genes as tRNA. Moreover, the annotation of both vB_PaeP_PS28 and vB_PaeM_PS3 genomes showed the absence of genes related to lysogenic cycle, antibiotic resistance and virulence encoding genes. These findings confirm that vB_PaeP_PS28 and vB_PaeM_PS3 are virulent phages and further support their suitability for therapeutic applications to combat P. aeruginosa infections. Finally, the influence of both vB_PaeP_PS28 and vB_PaeM_PS3 on P. aeruginosa pathogenesis was investigated in vivo using mice infection model. There was a significant reduction in the mortality of mice infected with P. aeruginosa and treated with phages; vB_PaeP_PS28 and vB_PaeM_PS3 as compared to bacteria-inoculated mice without phage treatment. Moreover, phage treatment effectively reduced bacterial colonization in the organs isolated from Pseudomonas-infected mice relative to those isolated from mice injected with bacteria alone. Interestingly, vB_PaeP_PS28 and vB_PaeM_PS3 have been found to be highly efficient in attenuation of P. aeruginosa virulence in mice, which further encourages their future application in phage therapy. It is worth mentioning that no detectable side effects or harmful signs were observed in mice upon phage treatment over the experiment course. In conclusion, current study highlights the influence of phage therapy as a promising tool for management P. aeruginosa infections. Isolated phages showed a pronounced bacteriolytic activity against P. aeruginosa as well as an efficient antibiofilm potential. In addition, the virulence capacity of P. aeruginosa in mice was significantly diminished upon treatment with isolated phages. Present data further support the efficiency of isolated phages as a novel candidate, either alone or to be incorporated in a phage cocktail therapy, to control P. aeruginosa infections.