BACKGROUND: Several metabolites released by fungal species are an essential source of biologically active natural substances. Gas chromatography high resolution time-of-flight mass spectrometry (GC-HRTOF-MS) is one of the techniques used in profiling the metabolites produced by microorganisms, including Talaromyces pinophilus. However, there is limited information regarding differential substrates' impacts on this fungal strain's metabolite profiling. This study examined the metabolite profile of T. pinophilus strain SPJ22 cultured on three different media, including solid czapek yeast extract agar (CYA), malt extract agar (MEA) and potato dextrose agar (PDA) using GC-HRTOF-MS. The mycelia including the media were plugged and dissolved in 5 different organic solvents with varying polarities viz.: acetonitrile, dichloromethane, hexane, 80% methanol and water, and extracts analysed on GC-HRTOF-MS. RESULTS: The study revealed the presence of different classes of metabolites, such as fatty acids (2.13%), amides (4.26%), alkanes (34.04%), furan (2.13%), ketones (4.26%), alcohols (14.89%), aromatic compounds (6.38%), and other miscellaneous compounds (17.02%). Significant metabolites such as acetic acid, 9-octadecenamide, undecanoic acid methyl ester, hydrazine, hexadecane, nonadecane, eicosane, and other compounds reported in this study have been widely documented to have plant growth promoting, antimicrobial, anti-inflammatory, antioxidant, and biofuel properties. Furthermore, T. pinophilus grown on PDA and MEA produced more than twice as many compounds as that grown on CYA. CONCLUSION: Thus, our result showed that the production of essential metabolites from T. pinophilus is substrate dependent, with many of these metabolites known to have beneficial characteristics, and as such, this organism can be utilised as a sustainable and natural source for these useful organic molecules.