Abstract: Thiochromanones and 1,3,4-thiadazoles as heterocyclic compounds have broad biological activities. In order to find novel compounds with antifungal activity, we synthesized a novel series of (E)-3-(((1,3,4-thiadiazol-2-yl) amino)methylene)-thiochroman-4-ones. Structures of these compounds were established by HR-MS, ¹H NMR, ¹³C NMR and 1D-noesy. All of the synthesized compounds were screened for antifungal activity by using an established agar double dilution method (plate method) against ten fungi species in vitro. Compound 5j showed significant inhibitory activity to Colletotrichum capsici, Rhizoctonia cerealis and Aspergillus niger compared with that of the positive control carbendazim. Compounds 5h exhibited better antifungal activity to Canidia albicans and Aspergillus funigatus than the positive control fluconazole, in which the minimum inhibition concentration can reach 8 μg·mL^-1 and 16 μg·mL^-1. Moreover, the molecular docking method was used to study the interaction mode of compound 5h and CYP51, and the results will be helpful for designing of CYP51 inhibitors in the future.