The environment significantly impacts the interaction between plants and pathogens, thus remarkably affecting crop disease occurrence. However, the detailed combined mechanisms of temperature and humidity influencing this interaction remain unclear. In this study, the interaction between tomato and Botrytis cinerea in various temperature and humidity conditions was analyzed by histological observation and a dual RNA-seq approach. Results showed that low humidity was not favorable for mycelial growth, resulting in infection failure. Both high and low temperatures at high humidity successfully inhibited pathogenic infection and disease incidence in the tomato plants, thus enhancing their resistance to B. cinerea. The high temperature and high humidity (HH) treatment induced the upregulation of light reaction genes, increased the net photosynthetic rate, and expanded the chloroplast morphology of infected tomatoes. The HH treatment also inhibited the expression of cell cycle-related genes of B. cinerea, interfered with conidial germination and mycelial growth, and damaged mycelial cell structure. Low temperature and high humidity (LH) treatment induced the expression of cell wall modification genes and remodeled the cell wall morphology of tomatoes in response to B. cinerea. In addition, the downregulated fungal catabolic genes and the abnormal increase in electron density of mycelial cells under LH treatment subsequently reduced the infection ability of B. cinerea. These results further explain the coupled effects of temperature and humidity on plant defenses and pathogen virulence, and provide a potential means to control gray mold.