Species within the Staphylococcus genus are important mastitis pathogens. Studies to describe virulence and antibiotic resistance as well as rapid techniques that permit analyses strains are needed. The aims were to identify and characterize Staphylococcus spp. isolated from mastitic milk, and to optimize multiplex polymerase chain reactions (PCR). Staphylococci previously isolated from milk of dairy cows with subclinical mastitis were analyzed. PCR was completed to amplify nuc, sodA, spa, agr locus, virulence factors, and antibiotic resistance genes. DNA sequencing of sodA and spa genes was performed and antibiograms were carried out on all isolates. In a group of 49 staphylococci, S. aureus was the most prevalent, followed by S. hyicus, S. xylosus, S. chromogenes. Following optimization of multiplex PCR, virulence factor genes were identified in the majority of isolates. The enterotoxin genes, seh and selx were highlighted. All hemolysin genes were detected in 28.6% of isolates. Antibiotic resistance was evaluated and the majority of isolates (69.4%) were resistant to penicillin. Among the genes encoding antibiotic resistance, mecA was identified, while two methicillin-resistant S. aureus were typed as spa type 605, agr type II, and one identified as SCCmec type IVa. The types t605 and agr II were detected in the majority of S. aureus assessed. The findings emphasized the importance of preventing Staphylococcus infection in dairy cows. Effective dairy herd management and information on milk quality are essential to prevent mastitis pathogens.