Multidrug-resistant Pseudomonas aeruginosa has emerged as an important opportunistic pathogen. This study elucidates the prevalence, antimicrobial resistance patterns, and biofilm formation of this pathogen in wound and urinary tract infections. Pseudomonas selective agar and MacConkey agar were used to analyze 432 clinical samples [wound swabs (210) and urine samples (222)] through selective culturing. Then, API 20E strips and the Vitek 2 Compact System were employed for the pathogen identification. Mueller-Hinton agar-based disk diffusion method revealed the antimicrobial susceptibility of Pseudomonas aeruginosa. The crystal violet staining method was adopted to investigate biofilm formation using microtiter plates. The virulence genes (toxA, algD, and phzS) were amplified via PCR. A low Pseudomonas aeruginosa prevalence (1.85%) was noted in analyzed samples. However, multidrug-resistance was noted in 65% of Pseudomonas aeruginosa isolates, whereas 25% isolates demonstrated extensive resistance. Despite the presence of the algD gene, biofilm formation was weak in most Pseudomonas aeruginosa isolates and did not correlate statistically with antimicrobial resistance. The virulence factors encoding genes (toxA, algD, phzS) were noted in most of the Pseudomonas aeruginosa isolates. The results highlighted the epidemiology of multidrug-resistant and virulent Pseudomonas aeruginosa in Saudi Arabia. The rising occurrence of Pseudomonas aeruginosa necessitates detailed elaboration on its virulence, prevalence, and antimicrobial susceptibility patterns in Saudi Arabia through molecular tools.