This study was aimed to isolate and identifyPseudomonas aeruginosa (P. aeruginosa) from 150 diseased broiler chickens in addition to 50 environmental swabs from water pipes and tanks to examine their susceptibility against some commonly used antimicrobial agents, in addition to detection of some virulence genes using Polymerase Chain Reaction (PCR) and evaluation of its ability to form biofilm in vitro. Clinically the affected chickens were subjected to postmortem examination (P.M) then samples from internal organs such as (liver, heart, lung, spleen, and intestine); tracheal and environmental swabs were collected and subjected to bacteriological examination and identification. Twenty P. aeruginosa isolates were recovered from the diseased chickens and environmental swabs with an incidence of (10%). Sixteen isolates were isolated from internal organs of 150 diseased chickens with an incidence of (10.66%); meanwhile 4 isolates were recoded from 50 water pipes and tanks swabs with a percentage of (8%). Antimicrobial sensitivity testing showed highly sensitivity to amikacin and colistin sulphate with percentages of (90%) for each of them. Doxycycline showed resistance with a percentage of (75%). PCR technique was a good tool for testing three virulence genes; pslA, pelAand fliC genes; the three genes were detected in of the examined samples with a percentage of (100%). A significant relationship between the existence of three virulence genes in the isolated P. aeruginosa and ability of biofilm formation (Slime producing ability) was reported in this study.