To develop more valuable and effective fungicide candidates, a novel series of 3,4-dichloroisothioxazole-based cycloalkylsulfonamides were synthesized and their structures were identified by 1H NMR, 13C NMR, MS and elemental analysis. Compound 3k was further confirmed by X-ray single crystal diffraction. The in vitro bioassay results demonstrated that the target compounds showed significant fungicidal activity on mycelial growth and spore germination of Botrytis cinerea. Especially, compound 3j, with prominent inhibition effect on mycelial with EC50 and EC80 values of 1.4 and 23.7 μg/mL respectively, was comparable to the selected commercial fungicide. Moreover, at 50 μg/mL, the inhibition rate of compound 3j on spore germination was recorded up to 89.7%. The further in vivo bioassay results indicated compound 3j continued to show high control effect on tomato leaves, flowers and fruit at 200 μg/mL, with control efficiencies of 94.3%, 89.3% and 91.9%, respectively. The structure-activity relationship showed that the compound with a five-membered ring possessed the best activity after the introduction of the active fragment of the 3,4-dichloroisothioxazole, provided a valuable idea for further creation of new fungicides.