A novel antifungalmycin N2 (3-methyl-3,5-amino-4-vinyl-2-pyrone, C6H7O2N) was previously discovered from Streptomyces sp. N2, which exerted a broad-spectrum antagonistic activity against phytopathogenic fungi. To provide comprehensive insights into the antagonistic mechanisms and biocontrol efficacy of antifungalmycin N2, the present work investigated the physiological responses of Rhizoctonia solani under interaction with antifungalmycin N2. First, the mycelial growth of R. solani was significantly inhibited by antifungalmycin N2 during liquid shake-flask culture. Morphological observations showed that the morphogenesis of R. solani was influenced by antifungalmycin N2, in which the hyphae became severely shriveled and flattened, irregularly folded and branched. Additionally, an obvious accumulation of reactive oxygen species (ROS) was detected in R. solani hyphae, indicating oxidative stress induced by antifungalmycin N2. Further results showed that chitinase activity and its hydrolytic N-acetylglucosamine were significantly accelerated by antifungalmycin N2, demonstrating the cell wall of R. solani was damaged. Interestingly, the enzymatic antioxidant activities of R. solani were significantly induced in response to a relatively low concentration of antifungalmycin N2 (1.44-5.77 µg/mL). However, all antioxidant enzymes became highly inactive when the antifungalmycin N2 was increased to 11.53 µg/mL, suggesting that the enzymatic antioxidant system in R. solani was probably collapsed by the oxidative stress beyond its acceptance scope. In conclusion, antifungalmycin N2 exerted its antagonistic activity by inducing both cell wall degradation and oxidative stress in R. solani, thus leading to fungal morphogenesis and autolysis. Meanwhile, R. solani could induce and activate its antioxidant enzymes as a defence response to the oxidative stress caused by antifungalmycin N2.