In this study, the inhibitory effect of Ganoderma formosanum mycelium extracts on tyrosinase, the central regulatory enzyme being responsible for cutaneous pigmentation, was investigated in both cell-free and cellular enzymatic systems, as well as in phenotype-based zebrafish model. Bioassay-guided purification indicated that the ethyl acetate fraction of G. fromosanum mycelium ethanolic extract (GFE-EA) demonstrated the highest inhibition toward cell-free tyrosinase (IC50 = 118.26 ± 13.34 ppm). The secreted and intracellular melanin of B16-F10 cells were reduced by GFE-EA through suppression of tyrosinase activity (IC50 = 102.27 ± 9.49 ppm) and its protein expression. Moreover, GFE-EA decreased surface pigmentation level of zebrafish via down-regulation of tyrosinase activity. Most of all, there is no significant difference in morphology and mortality between control and GFE-EA treated groups. Not only does GFE-EA exhibit similar depigmenting efficacy to kojic acid with lower dosage (approximately one-seventh of dose), but show less toxicity to zebrafish. It is worth noting that GFE-EA is extracted from mycelium, which subverts the general concept that mycelium lacks certain bioactivities possessed by fruit bodies. Altogether, it would appear that GFE-EA has great potential for application in the cosmetics industry.