This study conducted an experiment on the effects of microbial agents on the growth, development, and physiological and biochemical characteristics of tobacco seedlings using Guangdong Tobacco 97 as the material. The results showed that when mixed with microbial agents at a concentration of 1 500-3 000 g/hm² in the seedling substrate, the stem height, stem circumference, maximum leaf area, and dry matter accumulation in the roots and leaves of tobacco seedlings during the seedling stage increased by 1.56%-3.13%, 12.24%-14.29%, 9.49%-11.86%, 12.24%-16.33%, and 10.03%-13.84%, respectively, compared to the control group. The root vitality and leaf chlorophyll content increased by 9.53%-10.96% and 12.87%-16.18% respectively compared to the control group. At the same time, mixing microbial agents in the seedling substrate was beneficial for enhancing the activities of nitrate reductase (NR), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in tobacco leaves, reducing the malonaldehyde (MDA) content of tobacco seedlings, improving their stress resistance, and promoting their growth and development. The results of this experiment provide a reference for the application of microbial agents in the cultivation of strong tobacco seedlings.