<p>This work aims to provide a basis for the enhancement of fucoxanthin (FCX) and eicosapentaenoic acid (EPA) biosynthesis in the microalga <i>Phaeodactylum tricornutum</i> using metabolomics and computational biology. To achieve this, both targeted (UHPLC and GC-FID) and untargeted (FTIR and NMR) analyses were conducted throughout various stages of cell cultivation. Targeted analyses revealed that EPA concentrations peaked at the end of the logarithmic growth phase, while fucoxanthin levels remained consistent from the onset of this phase through to the stationary phase. Untargeted analyses provided metabolic profiles by correlating FTIR absorbance bands with functional groups. When combined with cultivation strategies designed to improve EPA and FCX content, the optimal time for harvesting cells was identified as the end of the logarithmic phase. NMR further highlighted potentially key metabolic pathways for optimizing EPA and FCX production in <i>Phaeodactylum tricornutum</i>, particularly those involved in glyoxylate and dicarboxylate metabolism.</p> Graphical Abstract <p></p>