The chemical and biological properties of carotenoids in the freshwater alga Oedogonium intermedium were investigated in this study. Carotenoids were extracted from the alga by dichloromethane and purified by saponification. The carotenoid content was determined both spectrometrically and by HPLC, the carotenoids identified by HPLC-PDA-APCI-IT-TOF-MS and the extracts analysed for several health-related bioactivities. The crude and saponified extracts contained 3,411.2±20.7 and 2,929.6±5.9µg carotenoids/g dry algal biomass, respectively. Seven major carotenoids were identified, namely neoxanthin, 9'-cis-neoxanthin, loroxanthin, violaxanthin, lutein, α-carotene and β-carotene, which were present in similar amounts in the alga. Both the crude and saponified carotenoid extracts exhibited significant antioxidant activities as well as potent inhibitory effects against several metabolically important enzymes including α-amylase, α-glucosidase, pancreatic lipase and hyaluronidase, but they were poor inhibitors of angiotensin converting enzyme (ACE). Oedogonium could be an important new source of carotenoids, specifically loroxanthin, which is lacking in terrestrial plants.