Pseudomonas aeruginosa causes urinary tract infections associated with catheters by forming biofilms on the surface of indwelling catheters. Therefore, controlling the spread of the bacteria is crucial to preventing its transmission in hospitals and the environment. Thus, our objective was to determine the antibiotic susceptibility profiles of twenty-five P. aeruginosa isolates from UTIs at the Medical Center of Trás-os-Montes and Alto Douro (CHTMAD). Biofilm formation and motility are also virulence factors studied in this work. Out of the twenty-five P. aeruginosa isolates, 16% exhibited multidrug resistance, being resistant to at least three classes of antibiotics. However, the isolates showed a high prevalence of susceptibility to amikacin and tobramycin. Resistance to carbapenem antibiotics, essential for treating infections when other antibiotics fail, was low in this study, Notably, 92% of the isolates demonstrated intermediate sensitivity to ciprofloxacin, raising concerns about its efficacy in controlling the disease. Genotypic analysis revealed the presence of various β-lactamase genes, with class B metallo-β-lactamases (MBLs) being the most common. The blaNDM, blaSPM, and blaVIM-VIM2 genes were detected in 16%, 60%, and 12% of the strains, respectively. The presence of these genes highlights the emerging threat of MBL-mediated resistance. Additionally, virulence gene analysis showed varying prevalence rates among the strains. The exoU gene, associated with cytotoxicity, was found in only one isolate, while other genes such as exoS, exoA, exoY, and exoT had a high prevalence. The toxA and lasB genes were present in all isolates, whereas the lasA gene was absent. The presence of various virulence genes suggests the potential of these strains to cause severe infections. This pathogen demonstrated proficiency in producing biofilms, as 92% of the isolates were found to be capable of doing so. Currently, antibiotic resistance is one of the most serious public health problems, as options become inadequate with the continued emergence and spread of multidrug-resistant strains, combined with the high rate of biofilm production and the ease of dissemination. In conclusion, this study provides insights into the antibiotic resistance and virulence profiles of P. aeruginosa strains isolated from human urine infections, highlighting the need for continued surveillance and appropriate therapeutic approaches.