Staphylococci can cause urinary tract infections (UTIs). These UTIs are among the significant causes of antibiotic resistance and the spread of antibiotic-resistant diseases. The current study is aimed at establishing a resistance profile and determining the pathogenicity ofStaphylococcusstrains isolated from UTI samples collected in Benin. For this purpose, urine samples (one hundred and seventy) that were collected from clinics and hospitals showed UTI in patients admitted/visited in Benin. The biochemical assay method was used to identifyStaphylococcusspp., and the disk diffusion method tested the antimicrobial susceptibility. The biofilm formation ability of the isolates ofStaphylococcusspp. was investigated by the colorimetric method. The presence ofmecA,edinB,edinC,cna,bbp, andebpgenes was examined by multiplex polymerase chain reaction (PCR). The results showed thatStaphylococcusspecies were identified in 15.29% of all infected individuals and that 58% of these strains formed biofilms. MostStaphylococcusstrains (80.76%) were isolated in female samples, and the age group below 30 years appeared to be the most affected, with a rate of 50%. AllStaphylococcusstrains isolated were 100% resistant to penicillin and oxacillin. The lowest resistance rates were seen with ciprofloxacin (30.8%), gentamicin, and amikacin (26.90%). Amikacin was the best antibiotic againstStaphylococcusstrains isolated from UTIs. The isolates carriedmecA(42.31%),bbp(19.23%), andebp(26.92%) genes in varying proportions. This study provides new information on the risks posed to the population by the overuse of antibiotics. In addition, it will play an essential role in restoring people's public health and controlling the spread of antibiotic resistance in urinary tract infections in Benin.