Rothmannia wittii, which is found in Binh Chau-Phuoc Buu Nature Reserve, Bung Rieng ward, Xuyen Moc District, Ba Ria-Vung Tau Province, Vietnam, is a rare species of Rubiaceae family. The present study aimed to determine the phytochemical composition and antimicrobial activity of the ethanolic extract of the R. wittii trunk. A total of 10 compounds were identified by liquid chromatography-mass spectrophotometry (LC-MS), including 1) benzene, 1-(1,5-dimethyl-4- hexenyl)-4-methyl, 2) diphenylsulfone, 3) 6β-hydroxy-10-O-acetylgenipin, 4) cinnamyl- cinnamate, 5) genipin, 6) megastigmatrienone, 7) ethyl p-methoxy-trans-cinnamate, 8) rothalazepane, 9) 1,3,6- trimethylpyrano[2,3-d ]imidazole-2,5(1H,3H)-dione, and 10) shanzhiol. The extract also exhibited an antibacterial activity against six bacterial strains: Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and Salmonella enteritidis, as determined via disk diffusion agar method.