Background and AimsMultidrug and extensive drug‐resistant Pseudomonas aeruginosa was extracted from burn patients referring to burn centers in southwest Iran so that biofilm generation and antibiotic resistance could be investigated.MethodsA specific primer was used to confirm all our considered 110 P. aeruginosa culture‐positive reports on 345 burn patients. The resistance of P. aeruginosa to seven antibiotics and Colistin with minimum inhibitory concentration (MIC) was assessed. Biofilm formation was assessed by the phenotypic study of specimens under Congo red agar and microtiter plate assays.ResultsOne hundred and 10 clinical P. aeruginosa isolates taken from burn wound infections were validated. Among P. aeruginosa isolates, Piperacillin, Ceftazidime, Maeropenem, Gentamycin, and Gatifloacin had the highest resistance to antibiotics, while Ticarcillin‐Clavulanic acid and Ceftolozane‐Tazobactam showed the least resistance. MICs were then evaluated via the E test. Seven isolates were resistant to colistin. Colistin reference MICs for multidrug‐resistant P. aeruginosa prevalence was 38%, while it was 22% for extensively drug‐resistant (XDR) P. aeruginosa. One P. aeruginosa was pandrug‐resistant (PDR). Under Congo red agar test, 66 isolates (67%) formed biofilms and black colonies, whereas 44 isolates (50%) had red colonies. In MTP, 76% formed biofilm. 40%, 32%, 21% of the isolates were strong, moderate, and weak biofilm formers, respectively, while 43% did not form biofilms.ConclusionThe P. aeruginosa resistance to antimicrobial agents has largely challenged the control of the infection. Accordingly, a higher resistance occurred when the isolates were transferred to the patients. Less than 50% P. aeruginosa samples generated strong biofilms. Consequently, hygienic measurements are essential to inhibit P. aeruginosa transmission to hospitalized patients.