<p>This study investigated the effects of different nitrogen (urea, nitrate, ammonium), iron (ferric citrate, ferric chloride, ferrous sulfate), and phosphorus (monopotassium phosphate, dipotassium phosphate, calcium phytate) sources on the growth, chlorophyll a, total lipids, and fatty acid composition of the diatom <i>Chaetoceros lorenzianus</i> using single-factor experiments. Among nitrogen sources, ammonium and urea promoted the highest biomass, while urea also maximized chlorophyll <i>a</i> content. Notably, ammonium significantly enhanced the yield of polyunsaturated fatty acids (PUFA) and eicosapentaenoic acid (EPA) compared to nitrate. For iron sources, ferric chloride (FeCl₃) was the most effective, improving growth, chlorophyll a, and the proportions of PUFA and EPA, without affecting total lipid content. Phosphorus sources did not influence growth significantly; however, calcium phytate and monopotassium phosphate increased total lipid yield, whereas dipotassium phosphate favored the accumulation of PUFA and EPA. This comparative analysis provides a foundational basis for selecting nutrient sources to tailor the biochemical profile of <i>C. lorenzianus</i> for potential applications.</p>