Staphylococcus aureus is causing high rates of morbidity and mortality in human. Stem cells secreted factors are safe and efficient alternative to stem cells in stem cells-based therapies. In this study, this study aimed to evaluate the antibacterial activity of factors secreted by human Wharton's jelly mesenchymal stem cells secretome (hWJ-MSCs) against Staphylococcus aureus, hWJ-MSCs werecultured and collected their secreted factors to be used for In vitro antibacterial screening against Staphylococcus aureus.Antibacterial activity, antibiofilm, and minimum inhibitory concentrations (MIC) of hWJ-MSC-S against Staphylococcus aureus were determined. The present data showed that hWJ-MSC secreted factors significantly inhibit the growth of two clinical isolate of Staphylococcus aureus. hWJ-MSC-secreted factors reduced the growth of Staphylococcus aureus by mor than 87% compared to un-treated control. MIC values were   4.68 µg/ml and 1.17 µg/ml for Staphylococcus aureus 76, and Staphylococcus aureus 105, respectively. The finding from this work could be utilized to develop an effective therapeutic approach to treat Staphylococcus aureus infection.