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 (EC50 ranging from 6.96-59.36??g/mL). It displayed the best inhibitory activity against Magnaporthe oryzae (EC50?=?6.96??g/mL), compared with azoxystrobin (EC50?=?12.04??g/mL), and significantly suppressed spore germination of M. oryzae with the inhibition rate reaching 100 percent (50??g/mL). The optical microscopy and scanning electron microscopy observations revealed that after treating M. oryzae mycelia with sanguinarine at 10??g/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.