Microemulsion-based animal oils, alone or associated with polymers have been extensively used in pharmacy, medicine and cosmetics, since the major lipid constituents of the oils show several biological activities. Despite showing antimicrobial activity, there are no reports in the literature regarding the effects of bullfrog oil on cytotoxic activity against tumor cells. The aim of the present study was to synthesize, characterise and evaluate the in vitro effects on melanoma cell line (B16F10) of bullfrog oil microemulsions associated or not with chitosan, surfactant and bullfrog oil (CSBO) and surfactant and bullfrog oil (SBO), respectively. The microemulsions were developed and their physical-chemical characteristics were evaluated by light microscopy, dynamic light scattering, atomic force microscopy and zeta potential. The microemulsions showed regular spherical shapes, high polydispersity and excellent (+82.2 ± 1.0 mV) to low (-16.0 ± 0.5 mV), colloidal stability. The systems significantly decreased the in vitro cell viability of melanoma skin cancer by up to 90.2% (CSBO) and 91.8% (SBO); while free bullfrog oil showed no effects. The results obtained from microemulsions of bullfrog oil indicate the potential of the microemulsions developed, alone or in combination with other chemotherapeutic agents, for future use in biomedical approaches aiming towards cancer therapy.