It is known that aminoglycoside antibiotics bind well to melanin biopolymer, but the relation between their affinity to melanin and ototoxicity is not well established. The aim of this work was to examine the impact of netilmicin on melanogenesis in cultured normal human melanocytes (HEMa-LP). The WST-1 assay was used to detect netilmicin cytotoxic effect. Netilmicin induced concentration-dependent loss in melanocytes viability. The value of EC50 was found to be - 75.0 mM. The analyzed antibiotic inhibited melanin biosynthesis in a concentration-dependent manner. Increasing the drug concentration resulted also in a decrease of cellular tyrosinase activity. Netilmicin-induced modulation of melanogenesis in melanocytes in vitro may explain a potential role of melanin and melanocytes in the mechanisms of aminoglycosides ototoxic effects in vivo.