The preformed antimicrobial compounds produced by maize, 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-one and its desmethoxy derivative 2,4-dihydroxy-2H-1,4-benzoxazin-3-one, are highly reactive benzoxazinoids that quickly degrade to the antimicrobials 6-methoxy-2-benzoxazolinone (MBOA) and 2-benzoxazolinone (BOA), respectively. Fusarium verticillioides (= F. moniliforme) is highly tolerant to MBOA and BOA and can actively transform these compounds to nontoxic metabolites. Eleven of 29 Fusarium species had some level of tolerance to MBOA and BOA; the most tolerant, in decreasing order, were F. verticillioides, F. subglutinans, F. cerealis (= F. crookwellense), and F. graminearum. The difference in tolerance among species was due to their ability to detoxify the antimicrobials. The limited number of species having tolerance suggested the potential utility of these compounds as biologically active agents for inclusion within a semiselective isolation medium. By replacing the pentachloronitrobenzene in Nash-Snyder medium with 1.0 mg of BOA per ml, we developed a medium that resulted in superior frequencies of isolation of F. verticillioides from corn while effectively suppressing competing fungi. Since the BOA medium provided consistent, quantitative results with reduced in vitro and taxonomic efforts, it should prove useful for surveys of F. verticillioides infection in field samples.