In this study, the design and synthesis of novel pyrido[1,2-a]pyrimidinone mesoionic derivatives incorporating dithioacetal structures were carried out. The three-dimensional quantitative structure−activity relationship (3D-QSAR) model was built according to the EC 50 values and directed the synthesis of compound A32. The biological activity test against Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) indicated that compound A32 showed good antibacterial activity with EC 50 values of 10.9 and 17.5 mg/L, which were lower than the EC 50 values of bismerthiazol (29.3 and 39.8 mg/L) and thiodiazole copper (64.8 and 78.1 mg/L). Furthermore, the in vivo antibacterial activity against bacterial leaf blight (BLB) and bacterial leaf streak (BLS) revealed that the protective activity of compound A32 was 43.9 and 41.7%, respectively, which was better than the protective activity of thiodiazole copper (40.6 and 35.0%). In addition, the protective activity against bacterial leaf blight of compound A32 was associated with the increasing rice defensive enzyme activity and the upregulation of proteins involved in oxidative phosphorylation. Moreover, compound A32 could upregulate the expression of complex I (nicotinamide adenine dinucleotide hydrogen (NADH) dehydrogenase) in the oxidative phosphorylation pathway, which was verified by complex I activity evaluation.