Infections by Mycobacterium spp. are responsible for thousands of deaths every year worldwide. Microbial resistance, toxic effects, and adverse consequences of conventional therapies bring forth the need to search for new therapeutic agents. The aim of this study was to determine the antimicrobial action of the molecule Rottlerin against Mycobacterium spp. The broth microdilution assay showed that Rottlerin inhibited the mycobacterial growth at concentrations ≤ 50 µg/mL (≤ 96.81 µM), and the lowest bactericidal concentration was observed against M. tuberculosis (25 µg/mL-48.40 µM). The cytotoxicity of Rottlerin was conducted in a epithelial cell culture and evaluated through 2,3-Bis-(2-Methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) colorimetric assay, revealing an IC