Abstract Cymoxanil (CYM) is a synthetic acetamide fungicide that has been widely used to combat downy mildew diseases in grapevine cultures and late blight diseases in tomato and potato caused by the oomycetes Plasmopara viticola and Phytophthora infestans , respectively. Despite its extensive application, the biochemical mode of action of CYM remains elusive. Previous reports indicate that CYM affects growth, DNA and RNA synthesis in Phytophthora and inhibits cell growth, biomass production and respiration rate in the well-characterized fungal model Saccharomyces cerevisiae . We therefore used this model to further characterize the effect of CYM on mitochondria. We found that CYM inhibits oxygen consumption in whole cells after 3 h of exposure, which persists over time. Using isolated mitochondria, we demonstrated that CYM specifically inhibits cytochrome c oxidase (C c O) activity during oxidative phosphorylation. Based on molecular docking algorithms, we propose that CYM acts by blocking the interaction of cytochrome c (cyt c ) with C c O, hampering electron transfer and inhibiting C c O catalytic activity. Although other targets cannot be excluded, our data offer valuable insights into the mode of action of CYM that can be instrumental to drive informed management of the use of this fungicide.