The aim of this study was to evaluate the effect of nordihydroguaiaretic acid (NDGA) on tumor bladder T24 cells. Bladder cancer T24 cells were cultured on Dulbecco's Modified Eagle Medium in presence of NDGA. Cell viability and apoptosis were evaluated after 24, 48 and 72 h by using fluorescein diacetate (FDA) and Alexa fluor 488 annexin-V/propidium iodide solution, respectively. To determine the mitochondrial effects of NDGA (0-24 h), reactive oxygen species (ROS) levels by dihydroethidium fluorescence, mitochondrial membrane potential (m) by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylimidacarbocyanine iodide (JC-1) dual fluorescence and cellular respiration states by high resolution respirometry were evaluated. It was found that NDGA reduced T24 cell viability after 72 h of incubation in a concentration-dependent manner and apoptosis increased at 48 h. Furthermore, 20 µM NDGA increased ROS levels, decreased m and promoted leak of respiration from mitochondrial respiratory chain in T24 cells which was associated to the death of tumor cells. Taken together these results suggested that antitumor effects of NDGA in T24 cells are related to its ability to induce mitochondrial alteration.