To thoroughly explore the application potential of the natural product 2,4,5-trimethoxybenzaldehyde in the field of agricultural fungicides, this study utilized 2,4,5-trimethoxybenzaldehyde, a major component of Acorus tatarinowii, as the starting material. A series of 27 chalcone derivatives containing acylpiperazine structures were designed and synthesized. The structures of these derivatives were characterized and confirmed using NMR and HRMS. The in vitro antibacterial activity of the target compounds was evaluated via turbidity method against plant pathogenic bacteria, including Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, and Xanthomonas axonopodis pv. citri. The results demonstrated that the majority of the target compounds exhibited significant inhibitory activity against Xanthomonas oryzae pv. oryzae. Notably, 1-(4-{2-oxo-2-[4-(thiophen-2-ylsulfonyl)piperazin-1-yl]ethoxy}phenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (designated as H5) displayed the most prominent antibacterial efficacy, with a half-maximal effective concentration (EC50) value of 22.50 μg/mL. This inhibitory potency was markedly superior to those of the reference agents bismerthiazol (47.78 μg/mL) and thiediazole copper (100.33 μg/mL), respectively. Furthermore, SEM and SYTO9/PI fluorescent staining further demonstrated that the target compound H5 exhibits significant bactericidal activity against Xanthomonas oryzae pv. oryzae.