This study aimed to investigate the in vitro antibacterial, antifungal, antibiofilm, antioxidant, and anticancer properties of isosteviol isolated from endangered medicinal plant Pittosporum tetraspermum. Pure compound was obtained and characterized by column chromatography followed by 1H NMR, 13C NMR, IR, and mass spectral analysis. The antimicrobial activities of the compound were assessed by the broth microdilution method and the antioxidant properties were determined using reducing ability assay, DPPH scavenging assay, hydroxyl radical scavenging activity, and superoxide radical scavenging assay. Anticancer study was evaluated by following MTT assay. Column purification and spectrocopical analysis lead to identifying isosteviol from the crude ethyl acetate extract. The compound exhibited significant activity against bacteria such as Staphylococcus epidermidis (125 µg/mL), Staphylococcus aureus (125 µg/mL), and Klebsiella pneumoniae (62.5 µg/mL). The MIC of the compound against Candida albicans, Aspergillus niger, and Trichophyton mentagrophytes was 62.5, 125, and 500 µg/mL, respectively. The compound showed comparatively better antibiofilm activity against E. coli, S. typhi, and P. aeruginosa. Furthermore, it exhibited good antioxidant properties. Anticancer properties of the compound against Vero and MCF7 cell lines were its advantage. Novel isosteviol would be useful to reduce the infectious diseases caused by pathogenic microorganisms or slow the progress of various oxidative stress-related diseases.