1,2-Benzisothiazol-3-one (BIT) exhibits limited antibacterial activity, prompting the search for new antibacterial drugs. In this study, BIT was used as the starting compound to synthesize 17 new N-substituted alkoxymethylbenzoisothiazolone derivatives through one-pot reaction method involving BIT, aldehyde, and alcohol. The reaction pathways were investigated using density functional theory (DFT), and the inhibitory activity of the new compounds against bacteria, fungi, and crop pathogens was evaluated. The results demonstrated that these compounds displayed strong inhibitory effects on Staphylococcus aureus and Candida albicans, with most compounds exhibiting minimum inhibitory concentrations (MIC) ranging from 1 mg/L to 4 mg/L. Compounds BIT-5, BIT-9, BIT-10, and BIT-11 exhibited high inhibitory activity against various bacteria and fungi tested, particularly against agricultural crop disease fungi such as Sclerotinia sclerotiorum, Fusarium pseudograminearum, Fusarium graminearum, and Gaeumannomyces gramims, with MIC values in the range of 5~10 mg/L, which has further research value.