BackgroundGroup BStreptococcus(GBS) is a causative agent of various infections in newborns, immunocompromised (especially diabetic) non-pregnant adults, and pregnant women. Antibiotic resistance profiling can provide insights into the use of antibiotic prophylaxis against potential GBS infections. Virulence factors are responsible for host–bacteria interactions, pathogenesis, and biofilm development strategies. The aim of this study was to determine the biofilm formation capacity, presence of virulence genes, and antibiotic susceptibility patterns of clinical GBS isolates.ResultsThe resistance rate was highest for penicillin (27%;n= 8 strains) among all the tested antibiotics, which indicates the emergence of penicillin resistance among GBS strains. The susceptibility rate was highest for ofloxacin (93%;n= 28), followed by azithromycin (90%;n= 27). Most GBS strains (70%;n= 21) were strong biofilm producers and the rest (30%;n= 9) were moderate biofilm producers. The most common virulence genes werecylE(97%),pavA(97%),cfb(93%), andlmb(90%). There was a negative association between having a strong biofilm formation phenotype and penicillin susceptibility, according to Spearman’s rank correlation analysis.ConclusionAbout a third of GBS strains exhibited penicillin resistance and there was a negative association between having a strong biofilm formation phenotype and penicillin susceptibility. Further, both the strong and moderate biofilm producers carried most of the virulence genes tested for, and the strong biofilm formation phenotype was not associated with the presence of any virulence genes.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-023-02877-y.