© 2022, Verduci International. All rights reserved.Objective: Malignant melanoma (MM) is the most aggressive skin cancer and treatment options are still limited in the late stages, generally accompanied by BRAF mutations. Usnic acid (UA), a well-known traditional lichen metabolite, has a promising and selective antitumoral activity. However, the effects of UA on MM cells with different genomic profiles in the BRAF aspect have not been investigated yet. In this study, we evaluated the effect of UA on BRAFV600E mutated-A2058 and wild-type MeWo cells. Materials and Methods: In the UA-treated cells, viability and cell death analysis were performed by using WST-1 and Annexin-V assays. Then, the death-related morphological changes were visualized by acridine orange(AO)/ethidium bromide (EB) staining. The cell cycle regulatory effect of UA was determined. Finally, time-dependent detection of acidic vesicular organelles (AVOs) was performed by live-cell imaging. Results: While MeWo viability significantly reduced to 53.8% and 28.6%, A2058 viability was detected as 61.3% and 50.3% at 50 and 100 µM UA for 48 h. Thus, MeWo cells were found to be more sensitive to UA. Annexin-V and morphological analysis results showed that UA triggered mainly a vacuole-dependent cell death by the formation of AVOs, instead of apoptosis, in the MM cells. This effect was prominent in A2058 compared to MeWo. UA also slightly triggered apoptosis in MeWo cells. Thus, the cell cycle regulatory effect of UA on MM cells changed based on the cell death type triggered. Conclusions: Our results suggest that UA exerts the cytotoxic effects on MM cells by inducing vacuole-dependent cell death, most probably autophagy, and the UA response of MM cells with a different genomic profile in the BRAF aspect varies.