Aims This study was conducted to determine the occurrence of staphylococci from cows with subclinical mastitis from independent herds in Algeria, and to characterize Staphylococcus aureus isolates. Methods and Results Quarter milk samples were collected separately, somatic cells were counted and samples with more than 200 000 somatic cells per ml were cultured on blood agar. Staphylococci isolates were identified by routine diagnostics, and S. aureus isolates were tested for antibiotic susceptibility by disk diffusion and microdilution. Congo red agar was used to detect biofilm formation and capsule synthesis was detected on serum soft agar (SSA). The S. aureus isolates were characterized by spa typing. DNA microarray analysis was performed to detect resistance and virulence genes. Overall, 40 center dot 0% (167/418) of the cows suffered from mastitis. In 63 center dot 5% (106/167) of the cows staphylococci were identified. Nine of the 106 Staphylococcus isolates (8 center dot 5%) were S. aureus. The coagulase-negative staphylococci belonged to 14 species. All S. aureus isolates were multiresistant and biofilm forming, with 66 center dot 67% of them showing diffuse colonies on SSA and belonged to CC97-agrI-cap5. Biofilm genes (icaA/C/D), 13 genes encoding for adhesion, six genes encoding proteases, 11 genes encoding superantigen like toxins were found. Genes conferring resistance to tetracycline (tet(K)), penicillin (blaZ/I/R) and macrolide-lincosamide-streptogramin B (erm(B), erm(A)) were also detected in the S. aureus from this study. Conclusions The current investigation provides a detailed molecular and biofilm formation ability of S. aureus involved in subclinical mastitis in Algeria and shows the wide distribution of adhesion and enterotoxin(-like) genes among S. aureus responsible for causing subclinical bovine mastitis. Significance and Impact of the Study These findings are valuable in tracking the evolution and genomic variation of S. aureus from bovine origin.