Marine protist Aurantiochytrium limacinum produces docosahexaenoic acid (DHA) as main polyunsaturated fatty acid (PUFA) and lacks any monounsaturated fatty acids (MUFA), while eicosapentaenoic acid (EPA) and MUFA’s are produced by Phaeodactylum tricornutum. The marine diatom P. tricornutum was co-cultured with A.limacinum to match the EPA:DHA ratio of fish oil. Modulation in initial cell density ratio overcame the dominance of A.limacinum during co-cultivation and led to regulated proliferation of both species. Media engineering with nitrate and glycerol concentration yielded 2:1 (56.44: 30.11) mg g⁻¹ and 1:1 (47.43: 49.61) mg g⁻¹ EPA: DHA ratio. The oil and biomass obtained from co-cultivation comprised of MUFA’s such as palmitoleic acid (2.65 mg g⁻¹) and oleic acid (1.25 mg g⁻¹) along with pigments like fucoxanthin (367.18 µg g⁻¹), β-carotene (8.98 µg g⁻¹) and astaxanthin (0.77 µg g⁻¹). Thus, co-cultivation of P. tricornutum with A. limacinum represented a unique strategy towards achieving desired fatty acid composition.