Basella rubra (Malabar spinach) is a commonly consumed green leafy vegetable in southern parts of India. The chemical composition, nutraceuticals characterization, squalene Nuclear Magnetic Resonance (NMR), in vitro antioxidant activities and cytotoxicity of B. rubra seed oil (33.08%) was investigated. Gas chromatography-mass spectrometry (GC/MS) analysis revealed the presence of palmitic (27.21 μmol%), oleic (33.83 μmol%) and linoleic acid (26.02 μmol%) with a total of 64.38 μmol% unsaturated fatty acids respectively. HPLC nutraceutical characterization showed a major constituent of gallic acid (11.23 mg%), γ-tocopherols (17.74 mg%), cycloartenylferulate (1.7 mg%), and squalene (1 g%). Squalene was further recovered (98%), purified (99.9%), and confirmed through 1H and 13C NMR. The in vitro antioxidant activities recorded by using 2,2-diphenyl-1-picrylhydrazyl (EC50 = 6 mg mL−1), ferric reducing antioxidant power (361.85 mM of Trolox Eq./100 g) and 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (EC50 = 56.19 mg mL−1) scavenging activity. In vitro cytotoxicity assessed on 3T3-L1 showed good cell survival without any toxicity (upto 400 μg mL−1). B. rubra seed oil has proven nutraceuticals and antioxidant potentials with least toxicity which can be recommended for functional foods applications.