Staphylococcus aureus and Pseudomonas aeruginosa are the major globally distributed pathogens, which causes chronic and recalcitrant infections due to their capacity to produce biofilms in large part. Biofilm production represents a survival strategy in these species, allowing them to endure environmental stress by altering their gene expression to match their own survival needs. In this study, we co-cultured different clinical isolates of S. aureus and P. aeruginosa as mono- and mixed-species biofilms in a full-strength Brain Heart Infusion Broth (BHI) and in a 1000-fold diluted Brain Heart Infusion Broth (BHI/1000) using Microtiter plate assay and determination of colony-forming units. Furthermore, the effect of starvation stress on the expression of pslA and fnbA genes of both species was investigated using RT-PCR. This work indicated that starvation stress significantly increased the biofilm biomass and bacterial density in all mono and mixed biofilm-producing strains. Interestingly, co-culture biofilms exhibited higher resistance to starvation as compared to monoculture. The current results also showed that the expression of fnbA and pslA genes was up-regulated under starvation stress in mono-and coculture biofilm. meanwhile, up-regulates of both genes in co-culture biofilm was significantly higher than mono- species biofilm.