Bipolaris sorokiniana synthesizes the 1,8-dihydroxynaphthalene (DHN) melanin via pentaketide pathway and promotes the development of aerial mycelia and conidia. A melanin biosynthesis inhibitor Tricyclazole (TCZ), brought changes when applied at 5-100 μg ml(-1) concentration in the colony morphology, radial growth, mycelia weight, melanin content, antioxidant enzymes (SOD and CAT) and extracellular hydrolytic enzymes (cellulase, pectinase, amylase and protease) in black, mixed and white isolates of B. sorokiniana. A significant alteration was recorded in antioxidant enzymes in black and mixed isolates; however, non-significant alteration was recorded in white isolate. Isolates of B. sorokiniana exposed to 100 µg ml(-1) TCZ showed significantly increased formation of superoxide radical (O 2 (·-) ) and hydrogen peroxide (H2O2)·H2O2 was detected significantly high in hyphae and conidia while, O 2 (·-) was found primarily in the conidia. Microscopic results suggest that TCZ damages not only the cell wall but also the cell membrane. The foliar application of TCZ (25, 50 and 100 µg ml(-1)) decreases the area under disease progress curve, lesion development and spore formation on barley leaves thereby reducing potential for the disease development. In conclusion TCZ influences the pathogenic ability by damaging the cell structure of hyphae and conidia and also alters the antioxidant enzyme levels in B. sorokiniana. TCZ may therefore, works against to pathogen for better management of spot blotch disease in barley infected with B. sorokiniana.