Abstract: As an important mean of biological control, microbial fertilizer is widely used in crop production. Tobacco seedling substrate environment is relatively simple, stable and controllable, which is beneficial to the colonization of microbes and the function of microbial fertilizer. In order to provide scientific basis and reference for the application of microbial fertilizer on tobacco, the study was conducted to evaluate the effect of two microbial fertilizers, Effective Microorganisms (EM) and Bacillus mucilaginosus, with different application rates (5%, 10% and 15%) and their combination on two tobacco varieties of‘Honghuadajinyuan’and‘Yunyan 85’. The results showed that adding different dosages of microbial fertilizer to tobacco seedbed improved the agronomic characters of the two tobacco varieties in maturity period, especially in the increase of plant height, distance between leaves, stem girth and leaf area index, which also improved the economic characters in yield, output value and the proportion of superior tobacco, especially in the improvement of the proportion of superior tobacco and output value. Overall,‘Honghuadajinyuan’added 15% EM microbial fertilizer or Bacillus mucilaginosus fertilizer, or‘Yunyan85’added 10% EM microbial fertilizer or Bacillus mucilaginosus fertilizer could gain better growth- promoting effects, and were recommended for popularization and application in production.