Prosthetic joint infections (PJI) are severe complications in Orthopedics, with Staphylococcus aureus and Staphylococcus epidermidis being the most commonly isolated pathogens. The variable antimicrobial susceptibility found in these microorganisms, along with the increasing number of methicillin-resistant strains, increases the difficulty of antibiotic selection and makes it necessary to perform individual susceptibility studies to select the optimal antibiotic treatment. The aim of this study was to evaluate the in vitro susceptibility pattern of 35 clinical strains isolated from PJI (17 S. aureus and 18 S. epidermidis) against rifampin, vancomycin, tygecicline, clindamycin, cotrimoxazole, cloxacillin, ciprofloxacin, daptomycin and fosfomycin. In vitro susceptibility assays were performed using the broth microdilution method and agar dilution for fosfomycin. MBC was also determined. Tygecicline and daptomycin showed the highest antimicrobial activity with low MIC 90 values, and no resistant strains were detected. On the other hand, ciprofloxacin and cloxacillin exhibited a poor antimicrobial effect with a high percentage of nonsusceptible strains in both species. Bactericidal activity rates revealed the bacteriostatic behavior of rifampin, tygecicline, cotrimoxazole, fosfomycin and clindamycin, whereas vancomycin and cloxacillin showed species-and strain-dependent behavior. Daptomycin and ciprofloxacin were observed to be efficient bactericidal agents against the tested strains. According to our data, rifampin, tigecycline, daptomycin and fosfomycin showed high in vitro activity against most staphylococcal strains isolated from the PJIs tested, although daptomycin seems to be the best alternative to vancomycin therapy.