Abstract Fungi is a well-known model used to study drug metabolism and its production in in vitro condition. We aim to screen the most efficient strain of Cunninghamella sp. among C. elegans, C. echinulata and C. blakesleeana for bromhexine metabolites production. We characterized the metabolites produced using various analytical tools and compared them with mammalian metabolites in Rat liver microsomes (RLM). The metabolites were collected by two-stage fermentation of bromhexine with different strains of Cunninghamella sp. followed by extraction. Analysis was done by thin layer chromatography, high performance thin layer chromatography, Fourier transform infrared spectroscopy, high performance liquid chromatography and Liquid chromatography–mass spectrometry. The role of Cytochrome P3A4 (CYP3A4) enzymes in bromhexine metabolism was studied. Fungal incubates were spiked with reference standard – clarithromycin to confirm the role of CYP3A4 enzyme in bromhexine metabolism. Three metabolites appeared at 4.7, 5.5 and 6.4 min retention time in HPLC. Metabolites produced by C. elegans and RLM were concluded to be similar based on their retention time, peak area and peak response of 30.05%, 21.06%, 1.34%, and 47.66% of three metabolites and bromhexine in HPLC. The role of CYP3A4 enzyme in metabolism of bromhexine and the presence of these enzymes in Cunninghamella species was confirmed due to absence of peaks at 4.7, 5.4 and 6.7 min when RLM were incubated with a CYP3A4 enzyme inhibitor – clarithromycin.