Staphylococcus saprophyticus is the second most frequent community-acquired causative agent of acute urinary tract infection (UTI). Some strains of S. saprophyticus can create biofilms, increasing their virulence. Once biofilms have been produced, antibiotic resistance is exacerbated. Hence, the aims of the present study were the study of biofilm formation, structure and antibiotic resistance in S. saprophyticus strains causing UTIs in women in Ahvaz, Iran. Overall, 43 S. saprophyticus isolates were recovered from UTIs. Antibiotic resistance pattern and the biofilm production and structure were determined using phenotypic methods. Most S. saprophyticus isolates were resistant to erythromycin, but all isolates were sensitive to linezolid and vancomycin. Fifty-eight per cent of S. saprophyticus were multidrug resistant. Twenty-one per cent of S. saprophyticus isolates harbored the mecA gene. Biofilm formation was observed in 65% of S. saprophyticus isolates and most had polysaccharide matrix. Our data indicate high rates of antibiotic resistance and the capability of biofilm formation among S. saprophyticus isolates. The emergence of antibiotic resistance in the management of UTIs is a serious public health issue. The findings of this study could be used to improve treatment plans to control UTIs. Consequently, increased awareness of the mechanisms underlying biofilm formation and the development of drug resistance will allow UTIs to be more efficiently controlled and treated.