The emergence of bacterial strains resistant to antibiotics in clinical use stimulates the research for new antimicrobials, especially in the case of multi-resistant strains such as MRSA (Methicillin Resistant Staphylococcus aureus). In the context of medicinal chemistry, the lapachol and derivatives are recognized by different pharmacological actions. This study evaluated the antistaphylococcal activity in vitro by disk diffusion method and determination of minimum inhibitory concentration (MIC) by microdilution of a lapachol semi-synthetic derivative, encoded as LSY. Cytotoxicity was assessed compared to the 2nd instar nauplii of Brine shrimp. The action of LSY against strains of Staphylococcus aureus (ATCC 25923) and MRSA (CCBH 5330) was verified by growth inhibition in the disk diffusion test and MIC values were determined to be 62.5 μg/mL for both microorganisms. The LC50 value corresponded to 14.23 μg/mL. Finally, the compound LSY showed a promising antistaphylococcal activity and relative toxicity in vitro by Brine shrimp bioassay. However, as the possibility of determining the toxicity in vivo should not be ruled out, better toxicological assessment of the molecule (LSY) is essential. A perspective for this study is the evaluation of tissue effects of compound topical use wounds in order that species of Staphylococcus can prevail in this type of injury. Thus, it is expected that the quinone evaluated (LSY) will be validated for therapeutic use in the future.