Therapy based on the induction of oxidative stress in melanoma cells may constitute a significant breakthrough in overcoming melanoma. Due to the fact that melanocytes are more sensitive to reactive oxygen species (ROS) than normal cells, it is possible to preferentially combat them by further induction of ROS. New derivatives of 4- fluorophenoxy and 4-chlorophenoxy acylthiosemicarbazide were designed and their antimelanoma potential was tested. A significant difference in biological activity, safety profile and lower cytotoxicity of derivatives with a fluorine atom were demonstrated. A decrease in the proportion of reduced glutathione (GSH) relative to oxidized glutathione (GSSG) and a disturbance of the redox potential were observed, which indicates the prooxidant properties of the tested derivatives. In addition, an increase in the expression level of the SOD2 gene was observed, and for the most active compounds against G-361 (IC50 for DD1 and DD4 is 107.62 μM and 89.09 μM, respectively) also a decrease in the expression of the CAT and GPX1 genes. FTIR microspectroscopy analysis was used for the first time. The obtained results revealed significant biochemical changes, including structural changes of proteins, disturbances of nucleic acid metabolism and increased lipid peroxidation, which indicates a mechanism induced by oxidative stress. The Langmuir monolayer model allowed for insight into the interactions of the tested compounds with the cell membrane, and in silico analysis showed a promising pharmacokinetic profile and low toxicity of the designed compounds. In the context of the conducted studies, 4-halophenoxyace- tylthiosemicarbazide derivatives constitute a promising group of potential antimelanoma agents, capable of disrupting the redox homeostasis of cancer cells and the integrity of the cell membrane.