Abstract: To compare disease resistance and phyllosphere microbial community structure between susceptible and resistant tobacco varieties, This study adopted resistance assessment combined with Illumina amplicon sequencing to evaluate the resistance of tobacco powdery mildew-susceptible variety K326 and its MLO mutant-resistant lines E167 and ZP43, as well as to characterize the composition and diversity of their phyllosphere microbial communities. The results showed that at 15 days post-inoculation, the disease indices of E167 and ZP43 were 0 ± 0 and 1.16 ± 0.14, respectively, significantly lower than that of K326 (96.6 ± 0.87). Phyllosphere microbial community analysis revealed that the dominant fungal genera in K326 were Golovinomyces (25.34%), Cladosporium (4.07%), and Penicillium (0.70%); in E167, Penicillium (37.44%), Papiliotrema (12.75%), and Trichoderma (8.46%); and in ZP43, Trichoderma (2.90%), Filobasidium (1.93%), and Penicillium (1.82%). In the MLO mutant-resistant lines (E167 and ZP43), the abundance of pathogenic fungi in the phyllosphere fungal community decreased, while the abundance of biocontrol agents significantly increased. The composition of dominant bacterial phyla and genera in the phyllosphere was similar across all varieties (lines), predominantly consisting of Pantoea, Enterobacter, and Pseudomonas, although their relative abundances varied. This study indicates that MLO gene modification can significantly promote the colonization of fungal genera including Trichoderma, Filobasidium, and Papiliotrema in the phyllosphere of E167 and ZP43, concurrently inhibit the colonization of pathogenic genus Golovinomyces, and enhance microbial community diversity, thereby improving the disease resistance of tobacco plants.