The high incidence of persistent multidrug resistant pseudomonal and staphylococcal infections is a global public health burden. Alternative strategies are required to deal with such issue including the use of drugs with anti-virulence activity (drug repurposing). The application of nanotechnology to develop advanced nanomaterials that target quorum sensing regulated virulence factors is an attractive approach. In the present study, a total of 20 clinical P. aeruginosa isolates in addition to 20 clinical S. aureus isolates were obtained from Microbiology and Immunology Department, Faculty of Pharmacy, Zagazig University. They were recovered from patients with burns, surgical wounds, respiratory tract, urinary tract and blood infections. Standard strains P. aeruginosa ATCC 27853 and S. aureus ATCC 6538 were used as controls. Isolates' identity was confirmed by conventional biochemical tests. The antibiotic susceptibility test was performed by using Kirby-Bauer disc diffusion method. All S. aureus and P. aeruginosa isolates were multi-drug resistant (MDR). P. aeruginosa isolates were investigated for the production of biofilm, pyocyanin, swarming motility and proteases. S. aureus isolates were investigated for the production of biofilm and staphyloxanthin. The most virulent P. aeruginosa and S. aureus isolates in addition to standard strains were selected for further work. Minimum inhibitory concentrations (MICs) of the tested agents; metformin (MET), metformin nanoemulsions (MET-NEs), silver metformin nanoemulsions (Ag-MET-NEs), silver nanoparticles (AgNPs), ascorbic acid (ASC) and ascorbic acid nanoemulsions (ASC-NEs) were determined using the broth microdilution method. There was no difference between the MICs of MET and MET-NEs against the tested bacteria. However, the MICs were markedly lowered upon using the combination of MET and AgNPs (Ag-MET-NEs) compared with either MET, MET-NEs or AgNPs suggesting synergy between MET and AgNPs that might be due to higher penetration. Similarly, there was no difference between the MICs of ASC and ASC-NEs against the tested bacteria. The virulence inhibitory activity of the tested agents was investigated at their sub-MICs (1/10) to avoid any effect of these agents on bacterial growth. The influence of the tested agents on the relative expression of the genes that regulates the virulence factors' production in the standard strains P. aeruginosa and S. aureus was assessed using qRT-PCR. The bacterial load was estimated in the presence and absence of sub-MICs of these agents using mice infection model. With regards to P. aeruginosa isolates, the phenotypic results showed that MET-NEs had the highest virulence inhibitory activity. However, concerning qRT-PCR results, all tested agents significantly decreased the expression of quorum sensing regulatory genes of P. aeruginosa; lasR, lasI, pqsA, fliC, exoS and pslA, with Ag-MET-NEs being the most potent one, however, this preparation failed to protect mice from P. aeruginosa pathogenesis. MET-NEs showed the highest protective activity against pseudomonal infection in vivo. Similarly, with regards to S. aureus isolates, the in vitro results showed a higher virulence inhibitory with both MET-NEs and Ag-MET-NEs treatment. However, genotypically, it was found that except for agrA and icaR genes that are upregulated, the tested agents significantly downregulated the expression of crtM, sigB, sarA and fnbA genes, with Ag-MET-NEs being the most efficient one. MET-NEs exhibited the highest protection against S. aureus infection in mice suggesting a role for AgNPs toxicity in these results. Ascorbic acid nano formulation exhibited better inhibition of P. aeruginosa biofilm and proteolytic activity. Both ascorbic acid and ascorbic acid nanoemulsions similarly inhibited swarming motility and showed variable effects against pyocyanin pigment. Ascorbic acid nano emulsion exhibited slightly higher anti-QS activity than ASC. In contrast to in vitro results, ASC-NEs failed to protect mice against pseudomonal infection. With regards to S. aureus isolates, the in vitro results showed that ASC-NEs showed stronger biofilm inhibitory activity than ASC, however, ASC-NEs showed slightly higher staphyloxanthin inhibition than ASC. The qRT-PCR results showed no significant difference between ASC and ASC-NEs. Bacterial treatment with both agents protected mice infected with these treated bacteria. These findings indicate the promising anti-virulence activity of nano formulations especially Ag-MET-NEs and ASC-NEs against MDR P. aeriginosa and S. aureus by inhibiting quorum sensing signaling system. In summary, further work is required to investigate the reason for variable effects of nano formulations against some virulence factors and the toxicity in mice models.