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The aim of this study is to determine the antibacterial activity of Piper betle L. leaf extract on inhibiting Staphylococcus aureus in conjunctivitis patient. This study follows a post-test only group experimental design. Antibacterial activities of five Piper betle L. extract concentrations (0.5%, 1%, 1.5%, 2%, 2.5%, and 3%) against Staphylococcus aureus were evaluated by agar well diffusion method. The negative control group (P-) was treated with standard 10% DMSO solution, the positive control group (P+) with ceftriaxone. The diameters of clear zone surrounding the well were analyzed with Kruskal-Wallis test and showed that in between 1% and 1.5%; 1.5% and 2%; 2% and 2.5%; 2.5% and 3% concentrations do not show a significant difference but in between 0.5% and 1%; 0.5% and 1.5%; 0.5% and 2%; 0.5% and 2.5%; 0.5% and 3%; 1% and 2%; 1% and 2.5%; 1% and 3%; 1.5% and 2.5%; 1.5% and 3%; 2% and 3% concentrations, Piper betle L. leaf extract shows a significant difference on inhibiting the growth of Staphylococcus aureus. In conclusion the results obtained show that that Piper betle L. leaf extract has a significant potential use as an antibacterial agent., Competing Interests: None., (AJCEI Copyright © 2020.)