Aim The current study aimed to establish a phenotypic and genotypic characterization record of a novel lytic bacteriophage (phage) against multidrug-resistant (MDR) Escherichia coli (E. coli) infections. Methods and Results Phenotypic characterization of the isolated phage included the assessment of phage morphology, host range, stability, and antibiofilm activity. The isolated phage vB_EcoM_ECO78 demonstrated a high lytic activity against MDR E. coli and E. coli serotypes O78: K80: H12 and O26: H11. Additionally, it showed a marked antibiofilm activity and high physical stability at a wide range of temperatures and pH. Genotypic investigations identified a double-stranded DNA genome of 165 912 base pairs (bp) spanning 258 open reading frames (ORFs), out of which 149 ORFs were identified and annotated. In vivo analysis further confirmed the therapeutic potential of vB_EcoM_ECO78 which effectively increased the survival of mice infected with MDR E. coli. Conclusion The isolated phage vB_EcoM_ECO78 exhibits considerable stability and antibiofilm activity against MDR E. coli isolates, supported by notable environmental fitness and in vivo antibacterial capability.