Cancer, diabetes, and antibiotic resistance of bacteria are serious problems in the field of health care and medicine. This research aims to determine the inhibition of DPP-IV in vitro, anticancer and antimicrobial properties of ethanol extract (TP) and its subfraction of ethyl ether extract (EE), chloroform (CF), n-butanol (BU), and water (WA) from the leaves of Rhinacanthus nasutus. In this study, the medicinal powder was extracted with 96% alcohol to get the ethanol extract. An amount of TP extract was dissolved in water before being extracted with increasing polarity solvents to yield ethyl ether (EE), chloroform (CF), n-butanol (BU), and water fractions (WA) from Rhinacanthus nasutus leaves. All extracts were tested in vitro for DPP-IV enzyme inhibition, anticancer, and antibacterial activities. The highest DPP-IV enzyme inhibitor efficacy was found in CF extract with an IC50 of 134.18 μg/mL. CF extract has the highest antibacterial resistance (the largest antibacterial circle diameter) and the highest MIC value of CF for bacterial Sta.aureus và B.subtilis: 625 μg/mL and 5000 μg/mL, respectively. In addition, the research results also evaluated the highest cytotoxic activity against breast cancer cells (MCF-7) in the ethyl ether fraction with inhibitory rate of 22.31 ± 1.85% (extract concentration at 100 µg/mL). The results of this study provide direction for further investigations into the application of Rhinacanthus nasutus as an antimicrobial and treatment for diabetes.