Understanding how drugs work in vivo is critical for drug design and for maximizing the potential of currently available drugs. 5-nitrofurans are a class of pro-drugs widely used to treat bacterial and trypanosome infections, but despite relative specificity 5-nitrofurans often cause serious toxic side-effects in people. Here, we use yeast, zebrafish and human in vitro systems to assess the biological activity of 5-nitrofurans, and identify a conserved interaction between aldehyde dehydrogenase (ALDH) 2 and 5-nitrofurans across these species. In addition, we show that the activity of nifurtimox, a 5-nitrofuran anti-trypanosome pro-drug, is dependent on zebrafish Aldh2 and that nifurtimox is a substrate for human ALDH2. This study reveals a conserved and biologically relevant ALDH2-5-nitrofuran interaction that may have important implications for managing the toxicity of 5nitrofuran treatment.