In continuation of our program to discover new potential antifungal agents, thirty-two neuchromenin analogues were synthesized and characterized by the spectroscopic analysis. By using the mycelium growth rate method, the target compounds were evaluated systematically for antifungal activities in vitro against six plant pathogenic fungi, and structure-activity relationships (SAR) were derived. Compounds 6b-c, and 6l showed obvious inhibition activity on each of the fungi at 50 μg/mL. For the corresponding fungi, 7 of the compounds showed average inhibition rates of >80% at 50 μg/mL; especially, compounds 6b, 6d-e, and 6i-l displayed more potent antifungal activity against A. solani than that of thiabendazole (a positive control). Moreover, compound 6c also exhibited good activity against C. lunata with EC50 values of 12.7 μg/mL, and the value was much superior to that of thiabendazole (EC50 = 59.7 μg/mL). SAR analysis showed that the presence of conjugated structure, bearing a C=C bond conjugated to the C=O group, obviously decreased the activity; the type and position of the substituted R5 significantly influenced the activity. Furthermore, the significantly bioactive compounds 6b-e, 6g, 6i and 6l showed very low toxicities against HL-7702, BEL-7402 and HCT-8 cells. Resistance development assay indicated that compounds 6b-e and 6l failed to induce the two tested strains of fungi to develop resistance. SEM analysis initially revealed that compound 6d may exert its antifungal effect by damaging fungal cell wall.