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The use of catheters and bladder catheters in hospitals can increase the risk of bacterial infections. This study aimed to identify the bacterial strains involved in catheter-related infections (CRI) in southern Benin hospitals. The study included 407 samples, including 95 catheter tip samples and 312 urine samples collected from bladder catheters from patients on the first day and 48 h after admission. The catheter tip samples were analyzed using traditional bacterial isolation and identification methods, while the urine samples were analyzed using VITEK-2. Antibiotic sensitivity was tested using the Kirby Bauer method, and virulence and resistance genes were detected through standard PCR. The results showed a predominance of Escherichia coli (53.5%), Klebsiella pneumoniae (23.3%), and Enterobacter aerogenes (7.0%) among Gram-negative bacilli, and coagulase-negative Staphylococcus as the most identified cocci. Bacterial susceptibility to antibiotics showed variable levels of resistance, with blaTEM being detected in 42.9% of identified bacterial species, followed by blaSHV (26.2%) and blaCTX-M-15 (16.7%). The blaNDM gene was only found in three identified bacterial strains, while vanA and vanB genes were detected in 3.2% of strains with a prevalence of 55% for the mecA gene. A prevalence of 18.8% for fimH was noted for the virulence genes. In conclusion, this study highlights the importance of following proper hygiene and aseptic practices during catheterization to effectively prevent CRIs. These findings should be used to improve interventions in hospitals and reduce healthcare-associated infections in developing countries.