Donkey milk, which is very similar to breast milk in terms of protein profile and lactose content, has been used for therapeutic purposes since ancient times. Different studies report its antimicrobial activity against an important human pathogen such as S. aureus. Therefore, in our study, we aimed to examine the antimicrobial efficacy of donkey milk against standard MRSA and MSSA strains in-vitro environment. In our study, in-vitro antimicrobial efficacy of donkey milk obtained from 53 different donkeys against S. aureus ATCC 43300 (MRSA) and S. aureus ATCC 29213 (MSSA) was investigated by broth microdilution method. Donkey milk mixed with TSB medium at different serial dilution rates was studied in 96 microwell plates. When the effectiveness of different concentrations of donkey milk added to TSB medium in vitro against ATCC MRSA and MSSA strains was examined, no suppression was observed, and growth was found to be higher than the control results after 24 hours of incubation at high milk concentrations. In conclusion, according to the results of our study, no antimicrobial effect of donkey milk was observed in-vitro against standard MRSA and MSSA strains. This situation may be caused by the fact that S. aureus strains may have different virulence abilities, as well as the differences in protein structure in milk between species.