Technical Abstract: Myrothecium verrucaria is a pathogen of several invasive weed species and is currently being evaluated for use as a bioherbicide. However, the fungus also produces macrocyclic trichothecene mycotoxins such as verucarins and roridins. The safety of this biological control agent would be improved if an inoculum could be produced without concomitant accumulation of trichothecenes. Sporulation and trichothecene production by M. verrucaria was evaluated on standard potato dextrose agar (PDA) and a series of defined media. Sporulation was supported on PDA and on agar media with nitrogen present in the nitrate form, but not on media with nitrogen only available as ammonium. Production of verucarin A ranged from 380 to 684µg plate-1 on PDA. Verrucarin A levels were over 1000 µg plate-1 after 15 days growth on a defined citrate and sucrose media with ammonium sulfate, but were below the detection limit with ammonium nitrate amended media. Cultures grown on molasses-ammended media produced more verracurin A compared to glucose or sucrose-based substrates. Roridin A was consistently observed on PDA, but frequently undetectable on defined substrates. Results support the hypothesis that accumulation of macrocyclic trichothecens by this fungus can be altered by manipulating carbon and nitrogen sources. Furthermore, the production of these mycotoxins can be uncoupled from sporulation, suggesting that the bioherbicide can be readily produced on solid substrates while simultaneously yielding conidia that are less threatening to worker safety.