It is estimated that one third of the world’s population is infected with M. tuberculosis, the causative agent of TB, and ranks alongside HIV in infectious disease mortality. Multi- and extensively-resistant infection to current frontline treatments is becoming more prevalent and, until recently, no new drugs had been developed for tuberculosis since the 1960s. The recently described flavin/deazaflavin oxidoreductases (FDORs) are potential drug targets since they are required for the persistent/latent M. tuberculosis infection. Although M. tuberculosis encodes fifteen F420 dependent FDORs, only a few have known activities with quinones, fatty acids or biliverdin. One of these enzymes, Rv0121c, appears to play an important role in persistence. We have solved the structure of the M. smegmatis homologue (MSMEG_6526; 70% amino acid identity), in complex with its cofactor, F420, at the active site. The structure reveals a dimer with the same split ß-barrel fold that is distinctive among the FDORs but with a smaller active site compared to other FDORs due to two larger loops flanking the site. One of these loops is highly dynamic, with open and closed conformations visible in the structure. Using this structure we have used high-throughput in silico docking to identify possible substrates to test for activity alongside a genetic knockout of Rv0121c in M. tuberculsosis, which has provided new insight into its physiological role and lead compounds for the development of inhibitors