Context:Hedyotis pilulifera (Pit.) T.N. Ninh (Rubiaceae) has been used in Vietnamese ethnomedicine; the methanol extract exhibited antibacterial activity in our preliminary screening.Objectives: In this study, compounds from H. pilulifera were isolated and their antibacterial activity in vitro was evaluated.Materials and methods: The aerial parts of H. pilulifera (1.4 kg) were extracted with MeOH, suspended in water and ethyl acetate extract was chromatographed on a silica gel column. The structures of isolated compounds were elucidated by the combination analyses of spectroscopy including 1D-, 2D-NMR, HRMS and in comparison with the reported NMR data in the literature. All isolated compounds were evaluated for inhibitory effect using the microdilution method toward Staphylococcus aureus, Bacillus subtilis and Mycobacterium smegmatis, and MIC values were determined.Results: Twenty compounds were isolated, including five triterpenoids, two steroids, two aromatic compounds, three fatty acids, one quinone derivative, one lignan glycoside, one ceramide and five glycolipids. Among these, oleanolic acid showed significant antibacterial activity against M. smegmatis with the MIC value of 2.5 μg/mL. Remarkably, rotungenic acid showed strong activity against S. aureus, B. subtilis, M. smegmatis with MIC values of 2.5, 2.5 and 1.25 μg/mL, respectively. Rotundic acid exhibited significant antibacterial activity against B. subtilis with the MIC value of 5 μg/mL. To the best of our knowledge, the antibacterial activity of rotungenic acid, stigmast-4-ene-3,6-dione and (2S,3S,4R,2′R)-2-(2′-hydroxytetracosanoylamino) octadecane-1,3,4-triol was reported for the first time.Conclusions: Oleanolic acid, rotungenic acid, and rotundic acid were considered to be useful for developing new antimicrobial therapeutic agents for human.