Pseudomonas aeruginosa is an important pathogen that is frequently associated with nosocomial infections. The goal of this work was to determine the relationship between the quorum sensing system (QS) and the production of virulence factors in P. aeruginosa. A number of 100 P. aeruginosa isolates were collected from various clinical sources from different Mansoura university hospitals in the period from April 2018 till April 2019. PCR screening of QS genes in the isolates was carried out including lasI, lasR, rhlI and rhlR. Thereafter, assay of the production of different virulence factors in the isolates was established including biofilm formation, pyocyanin production, protease production, lipase production, hemolysin production as well as swimming motility. Finally, statistical analysis of the data was performed to confirm the relationship between the QS and the production of virulence factors. Out of the 100 P. aeruginosa isolates, 27 clinical isolates were QS deficient. PCR analysis revealed that 8 isolates lacked lasR gene, 15 isolates lacked lasR and rhlR genes, 1 isolate lacked lasR and lasI genes, 2 isolates lacked lasR, lasI and rhlR genes and 1 isolate lacked rhlR, rhlI and lasR genes. There was a significant decrease observed in the production of pyocyanin, protease, lipase, hemolysin and biofilm formation as well as swimming motility in P. aeruginosa QS deficient isolates in comparison to non-QS deficient ones. There was a clear association between QS and virulence factors production in P. aeruginosa. This could open the door for novel promising targets for developing new therapeutic strategies against infections caused by this pathogen.