Pesticides are widely used to enhance food production on a global scale. However, little information is available on the effects of pesticide application on leaf physiology and phyllosphere bacterial communities of dioecious plants. Therefore, this study aimed to assess the impact of λ-cyhalothrin, a broad-spectrum pesticide, on leaf physiology and phyllosphere bacterial communities in the dioecious Populus cathayana. Physiological leaf traits such as photosynthetic apparatus (net photosynthetic rate (Pn), stomatal conductance (gs) and transpiration (E)) of males were significantly higher than those of females, independent of pesticide use. In contrast, pesticide application significantly reduced the photosynthetic apparatus for both sexes, and the reduction was greater in males relative to females. Also, pesticide application significantly increased peroxidase (POD) activity and malondialdehyde (MDA) content and maintained superoxide dismutase (SOD) activity and total chlorophyll content in leaves of males. The phyllosphere bacteria showed some conserved characteristics, in which, Simpson and Shannon diversity indices were not affected by sex or pesticide application. Phyllosphere bacterial community composition differed between females and males, indicating that intrinsic sex significantly shapes the phyllosphere bacterial community. However, pesticide application significantly increased the relative abundance of Actinobacteria but reduced the relative abundance of Proteobacteria. Principal component analysis showed associations between leaf physiology and specific bacterial taxa. For instance, Proteobacteria negatively correlated with leaf SOD activity and MDA content, while Actinobacteria showed an opposite pattern. Our study highlights sex-specific phyllosphere bacterial community composition and leaf physiological traits in dioecious plants.