<p>Mushrooms are rich in bioactive compounds like polysaccharides, phenolic acids, and terpenoids, known for their antioxidant, antimicrobial, and anticancer properties. Their bioactive profiles vary with cultivation, harvesting, and processing methods. Fermentation, a traditional method of food preservation, is known to enhance or alter the bioavailability and activity of bioactive compounds in several food sources. However, the impact of fermentation on the bioactive compounds of mushrooms remains underexplored. This study examines the effect of fermentation on the bioactive constituents of mushrooms using High-Performance Thin-Layer Chromatography (HPTLC) for qualitative analysis. The research focuses on selecting specific mushroom varieties, validating them against botanical reference materials, conducting fermentation with yeast and <i>Lactobacillus</i>, and comparing HPTLC fingerprinting of both raw and fermented mushroom powders. The results showed that fermentation significantly alters the fingerprinting of bioactive compounds in mushrooms, enhancing the levels of beneficial ones. The use of HPTLC as a profiling tool proved effective in distinguishing these changes, providing a reliable method for fingerprinting bioactive compounds in mushrooms. These findings suggested that the fermented mushrooms hold enhanced medicinal and nutritional potential over dried counterparts. Further research on specific bioactive could enable the development of functional foods or nutraceuticals with improved bioactivity.</p>