Abstract: A 7-year experiment was carried out to investigate the effects of no fertilizer (CK), chemical fertilizer (CF) and the mixture of manure and chemical fertilizer (MCF) on microorganisms in flue-cured tobacco grown soils. Plate count, microbial C and microbial N as well as phosphor lipid fatty acids (PLFAs) showed highest microorganisms in the soil treated with MCF, less with CF and lowest with CK. There were 20 (CK), 27 (CF) and 31 types of PLFAs (MCF) in soils, respectively. Fertilization increased the PLFAs contained in bacteria (1220C) and actinomycetes (10Me 17:0和10Me18:0). There were no obvious changes in fungal PLFAs (18:19c) in soils without fertilizer but a reduction with MCF. MCF increased both diversity and dominance indexes of soil microorganisms, indicating the improvement of soil environment because of abundant microbial groups and obvious dominant species. In addition, most of azotobacteria, phosphobacteria and potassium bacteria in soils belonged to Bacillus genus with the isolation rates ranged from 26% to 50%. Compared to CK, these three beneficial bacteria were increased by MCF but changed little by CF. The indexes of their diversity, homogeneity and dominance were higher in the soils under MCF treatment than CK. Taking into account of the numbers and groups of soil microorganisms, it is necessary to advocate MCF in fertilization practice.