INTRODUCTION: Breast cancer is one of the leading causes of cancer-related mortality in women worldwide. With the aim of developing more effective compounds against cancer and with fewer side effects than traditional chemotherapeutics, novel naphthoquinones (NfQs) with potential anticancer activity have been synthesized. OBJECTIVES: The aim of this study is to evaluate the action of the NfQ 3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione and its structural analogs NfQ1 (2-phenyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione) and NfQ2 (2-p-tolyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione) on the viability of MDA-MB-231 breast adenocarcinoma cell line, analyzing their ability to induce ROS production, mitochondrial dysfunction, and apoptosis. METHODOLOGY: MDA-MB-231 cells were incubated with NfQs (0 to 15 µM) for 24 h, and cytotoxic effects were quantified using the MTT assay. The CC50 was calculated from dose-response curves for each NfQ. Nuclear morphology were evaluated by fluorescence microscopy after staining cells with DAPI. Levels of ROS were assessed by flow cytometry using the probe DCFH-DA, and the production of O2- was evaluated using the NBT reduction technique. Mitochondrial membrane potential was assessed by flow cytometry using rhodamine-123. RESULTS: NfQs reduced the viability of MDA-MB-231 cells at concentrations above 0.5-1 µM after 24 h of culture, with similar CC50 values (p<0.01). Microscopic observation of DAPI-stained cells revealed condensed chromatin, membrane blebbing, and formation of apoptotic bodies, all events consistent with apoptosis. Incubation with NfQs increased levels of ROS and O2- compared to untreated cells (p<0.01). Additionally, NfQs induced alterations in mitochondrial membrane potential. CONCLUSIONS: NfQs exert cytotoxic effects on MDA-MB-231 cells by inducing oxidative stress and mitochondrial dysfunction, thereby promoting cellular apoptosis. These findings could contribute to improving current chemotherapy treatments.