Fertilization can be optimized and managed during the flue-cured tobacco growing period by studying the response of soil and microbial biomass stoichiometric characteristics to fertilization. In this study, we investigated the effect of compound fertilizers combined with microbial fertilizer treatments on the stoichiometric characteristics of the rhizosphere soil and the limitations of microbial resources during the flue-cured tobacco growing period. The results indicated that soil and microbial C:N:P varied greatly with the growing period. The effect of sampling time was usually greater than that of fertilization treatment, and microbial C:N:P did not vary with the soil resource stoichiometric ratio. The microbial metabolism of the tobacco-growing soil was limited by phosphorus after extending the growing period, and phosphorus limitation gradually increased from the root extension to the maturation periods but decreased at harvest. The rhizosphere soil microbial nitrogen and phosphorus limitations were mainly affected by soil water content, soil pH, microbial biomass carbon, and the ratio of microbial biomass carbon to microbial biomass phosphorus. Applying microbial fertilizer reduced phosphorus limitation. Therefore, applying microbial fertilizer regulated the limitation of microbial resources by affecting the soil and microbial biomass C:N:P in flue-cured tobacco rhizosphere soils.