Environmental strain Burkholderia sp. DNT mineralizes the xenobiotic compound 2,4-dinitrotoluene (DNT) owing to the catabolic dnt genes borne by plasmid DNT, but the process fails to promote significant growth. To investigate this lack of physiological return of such an otherwise complete metabolic route, cells were exposed to DNT under various growth conditions and the endogenous formation of reactive oxygen species (ROS) monitored in single bacteria. These tests revealed the buildup of a strong oxidative stress in the population exposed to DNT. By either curing the DNT plasmid or by overproducing the second activity of the biodegradation route (DntB) we could trace a large share of ROS production to the first reaction of the route, which is executed by the multicomponent dioxygenase encoded by the dntA gene cluster. Naphthalene, the ancestral substrate of the dioxygenase from which DntA has evolved, also caused significant ROS formation. That both the old and the new substrate brought about a considerable cellular stress was indicative of a still-evolving DntA enzyme which is neither optimal any longer for naphthalene nor entirely advantageous yet for growth of the host strain on DNT. We could associate endogenous production of ROS with likely error-prone repair mechanisms of DNA damage, and the ensuing stress-induced mutagenesis in cells exposed to DNT. It is thus plausible that the evolutionary roadmap for biodegradation of xenobiotic compounds like DNT was largely elicited by mutagenic oxidative stress caused by faulty reactions of precursor enzymes with novel but structurally related substrates-to-be.