Abstract The present study aimed to triggering of growth, lipid and fatty acids production of oceanic microalgae Tetraselmis sp. for biofuel production under mutagenic treatment with ethyl methanesulfonate (EMS). The maximum optical density (0.51 ± 0.02), cells concentration (138 ± 6.9 × 104 cells mL−1), biomass (0.63 ± 0.03 g L−1), chlorophyll ‘a’ (3.91 ± 0.19 mg L−1) were found in DKMK06 in first generation which was treated with EMS at 50 μmol mL−1 for 60 min. DKMK06 was exhibited the highest lipid productivity (48 ± 0.9%) during first generation and DKMK05 during second and third generations and percentages of lipid were 46.5 ± 1.1 and 41 ± 1.1%. The same trend has been observed in FAME contents and highest saturated and mono unsaturated fatty acid combinations were found in DKMK05 in 2nd generation (42.49%) followed by third generation (41.5%) which were higher than the first generation produced by DKMK06 (39.98%).