Microbial interactions are frequently categorized according to how they affect each population in a binary system. We aimed to determine the interaction between P . aeruginosa, S . aureus, and E . coli in-vitro. In this experimental hospitalized patients’ sputum, urine, and blood samples were used to collect a total of 90 clinical isolates for the study in Damanhour Medical National Institute, Behira, Egypt, followed by accurate identification and testing for antibiotic sensitivity. To examine the effect of the supernatant of P. aeruginosa on S. aureus and E. coli determined MIC using broth microdilution method. We also measured the activity of lasA protease by assessing the S. aureus cell lysis potential of P. aeruginosa culture supernatants. Extraction of pyocyanin was made to determine the change in the cell nature of S. aureus upon exposure to pyocyanin by using a scanning electron microscope and the shape of colonies on the culture media was determined. Finally, we detect lasA, operon phz, phzM, phzS and rhlAB genes for P. aeruginosa. P. aeruginosa showed a great impact on S. aureus isolates resistant to different antibiotics as it facilitates their killing and may drive the normal colonies of S. aureus into SCVs. The ability to form biofilm by S. aureus and E. coli decreased in the presence of Pseudomonas supernatant.