Background: Plumeria acutifolia (Apocynaceae) is an ornamental plant, used in the traditional medicine and known to have a variety of constituents as alkaloids, flavonoids, and iridoids. Extracts of this plant were proved to have antimicrobial and anticancer activities. Objectives: This research was conducted for the evaluation of the biological activities of P. acutifolia stem bark and isolation and structural elucidation of various chemical compounds from the biologically active fractions. Materials and Methods: Methanol extract of stem bark of P. acutifolia was successively fractionated with petroleum ether, dichloromethane, ethyl acetate, and n-butanol. The fractions were evaluated for their antimicrobial, cytotoxic, and antioxidant activities. Fractions with promising biological activities were subjected to chromatographic techniques for the isolation of compounds, followed by structural elucidation using several spectroscopic techniques. Results: P. acutifolia stem bark showed a significant antimicrobial activity, where the ethyl acetate fraction was active against Syncephalastrum racemosum (7.81 μ g/ml) and Escherichia coli (3.9 μ g/ml). The cytotoxic activity against HEPG-2, HCT-116, and MCF-7 cell lines was highest in the petroleum ether fraction, using concentrations of 1, 2.5, 5, and 10 μ l/ml. The antioxidant activity was concentration dependent; ethyl acetate fraction showed the most predominant effect, with an IC50of 197.1 μ g/ml. Five compounds were identified as narcissin (1), quercitrin (2), sweroside (3), gaertneroside (4), and plumieride (5). Conclusion: P. acutifolia was proved to have significant antimicrobial, cytotoxic, and antioxidant activities; the isolated compounds were flavonoids, iridoids, and secoiridoid, some of which were reported for the first time in genus Plumeria and/or family Apocynaceae.