The effects of biotic stresses on the contents of benzoxazinones (Bxs) were investigated in maize leaves. When the causal agent of southern corn leaf blight, Bipolaris maydis, was inoculated on the third leaf, the amount of 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA-Glc) increased, reaching a maximum level 48 h after inoculation. The inoculation of weakly pathogenic Curvularia lunata and non-pathogenic Alternaria alternata also resulted in accumulation of HDMBOA-Glc, and filtrates of the cultures of B. maydis, C. lunata and A. alternata also showed the accumulation of elicitor-active compounds by the fungi. Furthermore the infection of B. maydis induced formation of dark brown lesions, where most abundant Bx-related compound was 6-methoxy-2-benzoxazolinone (MBOA). The later is formed by degradation of DIMBOA and HDMBOA, whereas HDMBOA-Glc was most abundant in the surrounding green tissues. Among the Bx-related compounds, MBOA exhibited the strongest inhibition of the germination of the conidia and of the growth of germ tubes of B. maydis, C. lunata and A. alternata. In addition to fungal infection, the feeding by rice armyworm larvae resulted in HDMBOA-Glc accumulation. These findings are discussed in relation to the possible ecological relevance of the conversion of DIMBOA-Glc into HDMBOA-Glc.