Quorum sensing (QS) plays a very important role in virulence and biofilm formation of Pseudomonas aeruginosa. We tested the effect of the aqueous garlic extract (AGE) on QS regulated biofilm formation and on antimicrobial susceptibility profile of P. aeruginosa isolates. One hundred P. aeruginosa isolates from patients with nosocomial infections from different departments of Sohag University Hospitals were collected during the period from April 2016 to April 2017. These isolates were tested for antibiotic sensitivity by the disc diffusion method and were screened phenotypically for biofilm formation by the Congo Red Agar (CRA) method and Tissue Culture Plate (TCP) technique. The presence of LasI, LasR, Rh1I and Rh1R genes in biofilm forming P. aeruginosa isolates was tested by using polymerase chain reaction (PCR). Biofilm producing strains were investigated by exposure to sub-inhibitory concentration of AGE as a quorum-sensing inhibitory agent. P. aeruginosa showed the highest antibiotic resistance rate to Piperacillin (85%), followed by Ticarcillin-Clavulanate (84%), while the highest sensitivity was to Colistin (73%), followed by Polymyxin B (64%), and lastly to Meropenem (56%). Eighty % of the isolated samples were biofilm producers, and most of these were from ICU patients. Strains were found to have different distribution of individual QS genes. LasI gene was present in 74% of isolates, LasR gene was present in 58% of isolates, Rh1I gene was present in 43% of isolates and Rh1R gene was present in 36% of isolates. On exposure of biofilm producing strains to the AGE there was significant improvement of the antibiotic sensitivity profile and significant decrease of biofilm formation. This work highlighted the bioactivity of garlic extract, as a Quorum Sensing inhibitory agent, in effectively inhibiting biofilm formation and increasing the sensitivity of the pathogens to antibiotics.