Significance and Impact of the Study: Probiotics have been used in dairy products to promote human health and have the ability to metabolize drugs and other xenobiotics. Naphthoquinones, such as lapachol, are considered privileged scaffolds due to their high propensity to interact with biological targets. The present study is the first to demonstrate that probiotics are capable of converting lapachol into the most effective cytotoxic compound against a breast cancer cell line. The developed approach highlights the importance of probiotics to assess the effects of bacterial metabolism on drug performance and toxicity. AbstractProbiotics are currently added to a variety of functional foods to provide health benefits to the host and are commonly used by patients with gastrointestinal complaints or diseases. The therapeutic effects of lapachol continue to inspire studies to obtain derivatives with improved bioactivity and lower unwanted effects. Therefore, the general goal of this study was to show that probiotics are able to convert lapachol and are important to assess the effects of bacterial metabolism on drug performance and toxicity. The microbial transformations of lapachol were carried out by Bifidobacterium sp. and Lactobacillus acidophilus and different metabolites were produced in mixed and isolated cultures. The cytotoxic activities against breast cancer and normal fibroblast cell lines of the isolated metabolites (4a-hydroxy-2,2-dimethyl-5-oxo-2,3,4,4a,5,9b-hexahydroindeno [1,2-b]pyran-9b-carboxilic acid, a new metabolite produced by mixed culture and dehydro-a-lapachone produced by isolated cultures) were assessed and compared with those of lapachol. The new metabolite displayed a lower activity against a breast cancer cell line (IC 50 = 532Á7 lmol l À1 ) than lapacholdisplayed a higher activity than lapachol. The present study is the first to demonstrate that probiotics are capable of converting lapachol into the most effective cytotoxic compound against a breast cancer cell line.