SummaryDurian fruit rinds (Durio zibethinus) have increasingly attracted scientific interest due to their potent bioactive metabolites. In the present study, a comprehensive approach combining chromatographic techniques and bioassays was employed to elucidate the phytochemical constituents present in durian fruit rinds and their corresponding bioactivities. The crude ethanolic extract (CEE) and its fractions obtained through silica gel column chromatography (F1, F2, F3, and F4) were qualitatively and quantitatively analysed for their flavonoid profiles using thin‐layer chromatography, high‐performance liquid chromatography (HPLC), and liquid chromatography/mass spectrometry (LC/MS). Among the tested samples, fraction F2 exhibited the highest total flavonoid content (179.55 mg QE/g DW). TLC and HPLC analyses showed the presence of quercetin in all tested samples, with its content ranking in the order of F2 > F3 > F1 > F4 > CEE. The fractions exhibited inhibitory effects on albumin denaturation, protease activity, lipoxygenase (LOX), heat‐induced haemolysis, and xanthine oxidase (XO), surpassing those of the crude extract. These observed bioactivities correlated with the distribution of flavonoids and quercetin content in the samples. LC/MS analysis further showed the presence of flavonoids‐chalcone, flavonols, terpenoids, stilbene glycosides, anthocyanins, phenolics, and amino acids in fraction F2. These results determine the phytochemical profiles, anti‐inflammatory, and anti‐XO activities of bioactive fraction obtained from durian fruit rinds.