The aim of this study was to determine the content of fatty acids and amino acids profile in response to zinc and iron element in Pleurotus ostreatus. The influence of ZnSO₄, FeSO₄, ZnO and Fe₂O₃ at 80 µM on amino acid and fatty acid composition of P. ostreatus was investigated. Total amino acid was extracted using water: methanol: formic acid extraction solution and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fatty acid was extracted by lipid extraction and methylation procedure using acidic methanol: normal saline: hexane solution followed by gas chromatography-mass spectrometry (GC-MS). The main amino acids of P. ostreatus were arginine, glutamine, glutamic acid, alanine, serine, aspartic acid, lysine, threonine, histidine, valine and proline. Fe₂O₃ strongly lead a significant increase in essential and non-essential amino acids content of P. ostreatus. The most prominent fatty acids in P. ostreatus were linoleic acid, palmitic acid, oleic acid, stearic acid, pentadecanoic acid and heptadecanoic acid. ZnO strongly lead a significant increase in monounsaturated fatty acid (MUFA), omega-7 and omega-9 and significant decrease in polyunsaturated fatty acid (PUFA) and omega-6 fatty acids content of P. ostreatus. Iron is recommended for induction amino acid while zinc recommended for fatty acid production.