Phaeodactylum tricornutum is a marine diatom, and well-studied model of unicellular microalga. This diatom contains a wide range of high-value renewables (HVRs) with high commercial relevance owing to their importance in human nutrition and health. In this study, we screened P. tricornutum for biomass, eicosapentaenoic acid (EPA) and fucoxanthin production under photoautotrophic and mixotrophic condition with various substrate combinations. Results highlights that culture supplemented with glycerol and urea lead to enhanced biomass, biochemical and HVR production. Further continuous feeding of urea in glycerol supplemented medium results in an increase in biomass yield (0.77 g L-1) by ~ 2-fold. Additionally, continuous feeding of urea channelizes the carbon flux towards biosynthesis of fatty acids increasing FAME content by ~2-fold as compared to the control conditions. Overall EPA and fucoxanthin production was 27 mg L-1 and 11 mg L-1 (~2 & 4 fold) in urea fed cultures respectively. Present study demonstrates efficient valorization of cost-effective substrates such as glycerol and urea for the production of high-value renewables in P. tricornutum.