One of the most causative agents for many opportunistic diseases is the Pseudomonas aeruginosa which has a high percentage of multidrug resistance disease through construction of biofilm. The current study aimed for evaluating the correlation between quorum sensing genes (which is lasI gene) and biofilm formation. The biofilm construction and antibiotics susceptibility test were achieved for all the isolates under the study. The PCR and sequencing techniques were also carried out to detect the type of variation in lasI gene for each scheme of biofilm formation (weak, strong, and moderate). High antibiotic resistance was recorded among biofilm producing isolates. The genic pattern for the weak biofilm producer, which is isolate No 19, carries lysine instead of arginine in position 92 and aspartic acid instead of asparagine in position 115. Phylogenetic analysis indicated that the horizontal genetic transfer between isolates was very high. Finally, we conclude that the lasI gene does participate in biofilm construction of P. aeruginosa.