Isoquinoline alkaloids possess broad pharmacological activities. In this study, the antifungal activity of twelve isoquinoline alkaloids, including berberine (1), jatrorrhizine (2), coptisine (3), corydaline (4), tetrahydroberberine (5), chelidonine (6), dihydrosanguinarine (7), chelerythrine (8), sanguinarine (9), palmatine (10), tetrahydropalmatine (11) and columbamine (12) were evaluated against eight plant pathogenic fungi in vitro. All the tested compounds showed varying degrees of inhibition against the eight tested plant fungi. Among them, sanguinarine exhibited high antifungal activity (EC 50 ranging from 6.96–59.36 μg/mL). It displayed the best inhibitory activity against Magnaporthe oryzae (EC 50 = 6.96 μg/mL), compared with azoxystrobin (EC 50 = 12.04 μg/mL), and significantly suppressed spore germination of M. oryzae with the inhibition rate reaching 100% (50 μg/mL). The optical microscopy and scanning electron microscopy observations revealed that after treating M. oryzae mycelia with sanguinarine at 10 μg/mL, the mycelia appeared curved, collapsed and the cell membrane integrity was eventually damaged. Furthermore, the reactive oxygen species production, mitochondrial membrane potential and nuclear morphometry of mycelia had been changed, and the membrane function and cell proliferation of mycelia were destroyed. These results will enrich our insights into action mechanisms of antifungal activity of sanguinarine against M. oryzae. Unlabelled Image • Most of the tested isoquinoline alkaloids possessed antifungal activity against phytopathogenic fungi. • Sanguinarine demonstrated better antifungal activity and broader antifungal spectrum. • This result will enrich our insights into antifungal action mechanisms of sanguinarine against M. oryzae. • Sanguinarine may offer an effective and environmentally safe management strategy to control phytopathogenic fungi. [ABSTRACT FROM AUTHOR]