A novel macrolactam fungicide candidate (7B3) and a novel aza-macrolactone fungicide candidate (D1) were designed and synthesized, and the bioassay showed that both displayed excellent fungicidal activity against Rhizoctonia solani Kuhn. To elucidate the biochemical mode of action of the two compounds against R. solani and illustrate the similarities and differences of action mechanism resulting from subtle differences in structure of the two compounds, the effects of the two compounds on the ultrastructure of hyphae, electrolyte leakage, and respiration of mycelia cell suspension caused by 7B3 or D1 were studied. The results showed that the two compounds had very similar modes of action. Both induced irregular swelling of hyphae, vacuolation of cytoplasm, and thickening of cell wall. The conductivity of mycelia cell suspension increased in the presence of 7B3 or D1, which indicated that the two compounds had a similar effect on cell membrane permeability. In addition, both 7B3 and D1 were insufficient in inhibiting the respiration of mycelia.