Mitochondrial dysfunction has been recognized as an essential contributor to many human diseases including neurodegenerative disorders. However, the exact pathological role of mitochondrial dysfunction, especially in mitochondrial reactive oxygen species-associated oxidative stress, remains elusive, partially due to the lack of chemical probes with well-defined mechanisms of action. Herein, we describe the characterization and discovery of a rationally designed small molecule ZCM-I-1 as a selective modulator of the production of reactive oxygen species from mitochondrial complex I that does not alter mitochondrial membrane potential and bioenergetics. Chemical biology studies employing photoaffinity probes derived from ZCM-I-1 demonstrated its novel mechanism of action of modulating complex I via interactions with the flavin mononucleotide site, proximal in the reaction pathway within complex I.