Soil amendment is an important strategy to improve soil quality and crop yield. During 2014–2019, we investigated the effects of tobacco straw return with lime on soil nutrients, soil microbial community structure, tobacco leaf yield and quality in southern Anhui, China. A field experiment was conducted in four treatments: straw removed (CK), straw return (St), straw return with dolomite (St + D), and straw return with lime (St + L). Results showed that after five years of continuous treatment, the St + L significantly increased the soil pH by 16.9%, and the contents of soil alkaline nitrogen (N) and available potassium (K) by 17.2% and 23.0%, respectively, compared with the CK. Moreover, the St + L significantly increased tobacco leaf yield (24.0%) and the appearance (9.1%) and sensory (5.9%) quality of flue-cured tobacco leaves. The addition of soil conditioners (straw, dolomite, and lime) resulted in an increase in both the total reads and effective sequences of soil microorganisms. Bacterial diversity was found to be more sensitive to changes in the external environment compared to soil fungi. The application of soil amendments (lime and straw) has been shown to promote the growth of beneficial microorganisms in the soil. Additionally, bacterial species face greater competition and limited availability of resources for survival compared to fungi. Various analytical techniques, such as RDA, correlation analysis, and network analysis, were employed to investigate this finding: soil microorganisms are significantly influenced by the presence of AK, AN and pH contents. These findings can provide an effective method for improving the quality of flue-cured tobacco leaves and guiding the amelioration of acidic soil in regions where tobacco-rice rotation is practiced.