Spirolobium cambodianum Baill., belonging to Apocynaceae family, was used as a traditional medicine to treat various diseases. This study is the first report on antioxidant activities and chemical composition of S. cambodianum Baill. ethanolic leaf extract. The results showed that ethanolic extract had potent in vitro antioxidant activities in DPPH, ABTS•+ assays (the low concentrations of IC50 were 19.51 ± 0.78 µg/mL and 4.89 ± 0.07 µg/mL, respectively), and in RP, TAC methods (the Abs0.5 were 20.80 ± 0.26 µg/mL and 36.15 ± 1.03 µg/mL). Moreover, investigating in vivo antioxidant activity under oxidative stress conditions caused by paraquat and H2O2 using Drosophila melanogaster showed that flies were raised in the medium supplemented with a concentration of extract (1 mg/mL) lived longer than standard medium. Notably, the chemical investigation of S. cambodianum extract, ten identified compounds were reported for the first time from this plant and from the genus Spirolobium, including 27‐p‐Z‐coumaroyloxyursolic acid (1), lupeol (2), p‐hydroxybenzoic acid (3), ursolic acid (4), tectoquinone (5), daucosterol (6), asiatic acid (7), naringenin (8), 1‐O‐β‐D‐glucopyranosyl‐(2S,3S,4R,9Z)‐2‐[(2′R)‐2′‐hydroxytetracosanoylamino]‐octadec‐9‐en‐1,3,4‐triol (9), quercetin (10). This study suggests that the antioxidant efficacy of S. cambodianum leaf extract and its constituents can be explored as a potential antioxidant agent to treat various diseases, especially oxidative stress.