Abstract <p>In this study, a series of novel 1,3,4-oxadiazole thioether derivatives incorporating a methylsulfonyl moiety were synthesized and their structures were characterized by ¹H, ¹³C NMR, and HRMS. Bioassay results demonstrated that the target compounds exhibited moderate to good in vitro antibacterial activities against <i>Xanthomonas oryzae</i> pv. <i>oryzae</i> (<i>Xoo</i>), <i>Xanthomonas axonopodis</i> pv. <i>citri</i> (<i>Xac</i>), and <i>Wickerhamomyces anomalus</i> (<i>Wa</i>). Notably, 2-[(4-fluorobenzyl)thio]-5-[4-(methylsulfonyl)phenyl]-1,3,4-oxadiazole showed excellent antibacterial activities against these pathogens at concentrations of 200 and 100 μg/mL, with inhibition rates of 90 and 76% for <i>Xoo</i>, 84 and 64% for <i>Xac</i>, and 96 and 81% for <i>Wa</i>, respectively, which were even better than thiodiazole copper. However, the target compounds exhibited certain in vitro antifungal activities against <i>Penicillium sumatraense</i> (<i>Ps</i>) and <i>Aspergillus fumigatus</i> (<i>Af</i>) at 50 μg/mL, with the inhibition rates of 25–46 and 18–42%, respectively, which were lower than those of prochloraz. To the best of our knowledge, it is the first report on the synthesis and bioactivity evaluation of this series of 1,3,4-oxadiazole thioether derivatives incorporating a methylsulfonyl moiety.</p>