Moringa oleifera Lam is an economically and medicinally important plant. However, its essential oil characterization has been limited to one-dimensional gas chromatography and mass spectrometry. This study identified secondary metabolite composition and variation in M. oleifera seed oil through two-dimensional gas chromatography with time of flight mass spectrometry and their associated bioactivity. GC×GC TOF MS analysis of M. oleifera seed oil was performed on an Agilent 7890 Gas chromatograph equipped with Pegasus 2D GC-TOFMS. About 1µl of the sample (dissolved in n-Hexane) was injected into the system, and the carrier gas was Helium. Identification was made using ChromaTOF software with reference to the NIST library. A total of 2000 phytoconstituents were obtained, of which 236 were identified using the NIST mass spectral values. Total constituents were classified into alkanes (64), alkenes (11), aldehydes (7), alcohol (10), acids (18), acid esters (70), Ketones (10), benzenoids (10), Monoterpenoids (1), olefins (6), Phenols (1), an alkaloid (1), triterpenoid (4), diterpenoid (1), sesquiterpenoid (2), tocopherol (2), and Others (18). Based on area percentage, fatty acids and their derivatives were predominant. The major constituents were Erucic acid (9.10%), trans-13-Octadecenoic acid (6.06%), Triethyl citrate (5.15%), Bis-(3,5,5-trimethylhexyl) phthalate (4.94%). This study reports a detailed metabolic profiling of M. oleifera seeds, which opens up the possibility of identifying and decoding specific bioactivities leading to novel drug discovery in the future.