The research entitled, “ANTIBACTERIAL PROPERTIES OF MAKAHIYA ROOTS (Mimosa pudica) AGAINST Staphylococcus aureus In vitro” was a quantitative experimental study conducted to examine the ability of Mimosa pudica roots in inhibiting the growth of S. aureus. Phytochemical testing was conducted to determine the contents of the ethanolic extracts of M. pudica roots and found the presence of saponins, tannins, alkaloids, flavonoids, terpenoids, glycosides, proteins, carbohydrates, and phenols. The study employed the agar well diffusion method to determine the capability of M. pudica roots to inhibit the growth of the mentioned bacteria. Results of 100% extract concentration produced a zone of inhibition of 25.33 mm, while 50% produced a zone of inhibition of 21.00 mm. A zone of inhibition of 17.00 mm was measured for 25% and 14.67 mm for 12.5% concentration. The results of the agar well diffusion method showed the zone of inhibition increased as the concentration of the ethanolic extracts of M. pudica roots increased. Also, a significant difference was found between the mean zone of inhibition of the varying extract concentrtaions. The conclusive impact of the results of the study can offer new approaches and methods in traditional medicine, offering cost-efficient and effective ways that can inhibit and prevent the growth of bacteria.