A series of novel 1,3,5-thiadiazine-2-thione derivatives containing a 1,3,4-thiadiazole group was designed and synthesized. The structures of all the compounds were well characterized using H NMR, C NMR and high-resolution mass spectrometer, and further confirmed by the X-ray diffraction analysis of . The antimicrobial activities of all the target compounds against , , and were evaluated. The in vitro antimicrobial bioassays indicated that some title compounds exhibited noteworthy antimicrobial effects against the above strains. Notably, the compound -(5-(ethylthio)-1,3,4-thiadiazol-2-yl)-2-(5-methyl-6-thioxo-1,3,5-thiadiazinan-3-yl)acetamide () displayed obvious antibacterial effects against and at 100 μg/mL with the inhibition rates of 30% and 56%, respectively, which was better than the commercial bactericide thiodiazole-copper. In addition, the anti- EC value of was 33.70 μg/mL, which was more effective than that of the commercial fungicide hymexazol (67.10 μg/mL). It was found that the substitutes in the 1,3,5-thiadiazine-2-thione and the 1,3,4-thiadiazole rings played a vital role in the antimicrobial activities of the title compounds. More active title compounds against phytopathogenic microorganisms might be obtained via further structural modification.