BackgroundStaphylococcus spp. is one of the most causative agents of urinary tract infections (UTIs). This study aimed to investigate the antibiotic resistance profile and the virulence factors, including the biofilm formation ability ofStaphylococcus spp.isolates from urine.MethodologyThe agar disk diffusion method was used to test the susceptibility ofStaphylococcusisolates to ten antibiotics. The biofilm formation ability was determined using the safranin microplate-based method, and the phospholipase, esterase, and hemolysin activities were assessed by the agar plate method.ResultsDuring the study period, a prevalence of 18.12% of urinary tract infections caused by the identified Staphylococci was obtained. All the isolatedStaphylococcus aureusandS. epidermidiswere resistant to cefazolin. Multi-drug resistance (MDR) was recorded in 80.01%, 81.49%, and 76.20% ofS. aureus,S. epidermidis,andS. saprophyticusisolates, respectively. Most of the isolates were moderate biofilm formers, while 44.44%, 31.75%, and 30.16% were positive for phospholipase, esterase, and hemolysin activities, respectively. No relevant correlations were observed between the ability of biofilm formation and the resistance to antibiotics or the expression of virulence factors investigated.ConclusionThis study shows thatStaphylococcus spp.isolates from patients with clinical manifestations of UTIs expressed a high degree of virulence factors, including the ability of biofilm formation, and exhibited multi-drug resistance to the majority of antimicrobials commonly used for the treatment of Staphylococcal infections.