Substrate cultivation has become the mainstay of most vegetable cultivation. Many studies have shown that microbial agents can promote the release of nutrients in the cultivation medium and thus meet the needs of crop growth and development. In this study, the effects of six microbial agents on the growth of tomato plants and fruit development under substrate cultivation conditions were investigated using ‘Provence’ tomato as the material, and the physical and chemical properties of the substrates were measured and analyzed. It was found that the six microbial agents used in this study could promote the nutrients in the substrate, the growth and development of tomato plants, and the improvement of fruit yield and quality to different degrees. In this study, the two microbial agents, Bacillus subtilis and Bacillus cereus, can promote the release of nutrients in the substrate, the growth of plants, and the conversion of substances in the fruits, and realize the quality of the quality of the yield, and the results of this study can provide a technical guide for the cultivation of tomatoes in an efficient and high-quality substrate.