Iron/siderophore uptake may play an important role in the biofilm formation and secretion of extracellular proteins in Pseudomonas aeruginosa isolates. In the present study, the role of siderophores, heme, and iron regulatory genes in the virulence of Pseudomonas aeruginosa isolates collected from wound infection was investigated. Three hundred eighty-four (384) swab samples were collected from wound infection and identified by phenotypic methods. The quantitative real-time PCR (qRT-PCR) method was evaluated for the gene expressions study. Multi-locus sequence typing (MLST) was used to screen unique sequence types (ST) and clonal complexes (CC). Fifty-five (55) P. aeruginosa isolates were detected in all swab samples. Also, 38 (69.1%) isolates formed biofilm. The prevalence of virulence factor genes was as follows: plcN (67.2%), exoY (70.9%), exoA (60.0%), phzM (58.1%), plcH (50.9%), lasB (36.3%), aprA (69.1%), lasA (34.5%), nanI (74.5%), exoU (70.9%), exoS (60.0%), exoT (63.6%) and algD (65.4%). According to qRT-PCR, genes regulating iron uptake were highly expressed in the toxigenic isolate. The highest expressions levels were observed for hemO, hasR, and pvdA genes in the biofilm-forming isolates. The MLST data confirmed a high prevalence of ST1, ST111, and ST235, with six, five, and 12 clusters, respectively. ST235 and ST1 were the most present among the biofilm-forming and toxigenic strains. Also, the nuoD gene with 54 and guaA with 19 showed the highest and lowest number of unique alleles. We demonstrated that iron/siderophore uptake is sufficient for biofilm formation and an increase in the pathogenesis of P. aeruginosa. These results suggest that the iron/siderophore uptake system may alter the MLST types of P. aeruginosa and predispose to bacterial pathogenesis in wound infections.