Abstract: Compounds bearing structurally unique isoquinolin-1(2H)-one scaffold were found to be widely distributed in various organisms and they exhibited a broad spectrum of biological activities. In the present study, 22 derivatives of the isoquinolin-1(2H)-one scaffold were synthesized by Castagnoli-Cushman reaction and esterification reaction and their antifungal activity was evaluated against 7 phytopathogens. The in vitro bioassay results indicated that the mycelial growth inhibition rates of compounds 5a-5k and 6a-6f were higher than 80% against Sclerotinia sclerotiorum at the concentration of 100 μg/mL. Compound 5k was the most potent activity with the EC₅₀ value of 5.8 μg/mL, which was lower than the commercial boscalid (EC₅₀ = 0.094 μg/mL). The structure-activity relationship analysis revealed that the phenyl on N atom site is superior to the alkyl, and the introduction of different substituents to the phenyl group could improve the antifungal activity, which was subject to the positional selectivity and the number of substituents on the phenyl group. However, introducing different substituents at C3 phenyl and C4 carboxyl group were unfavorable for the activity. The preventive control efficacy of 5k was 94.6% at 500 μg/mL, which was comparable to that of boscalid at 10 μg/mL (95.8%). Taken together, these results could provide some important cues for further optimization of tetrahydroisoquinolone-4-carboxylic acid derivatives.