Abstract: In order to alleviate the obstacle of continuous tobacco plantation, a field experiment was conducted to analyze the effects of biofumigation with marigold stalks on the prokaryotic microbial community in tobacco rhizosphere soil. The results indicated that the application of marigold stalks at the rate of 750-3 000 kg/ha increased the amount of bacteria and actinomycetes in rhizosphere soil. The amounts reached the maximum at the rate of 1 500 kg/ha. Moreover, biofumigation with marigold stalks at 1 500 kg/ha could significantly enhance the diversity of prokaryotic microbial community. After tobacco plant transplanting for 50 d, biofumigation raised the Shannon, Sobs and Chao 1 indexes of soil prokaryotic microbial community by 6.09%, 11.81% and 11.27% respectively. The structure of prokaryotic microbial community was also improved after biofumigation with marigold stalks at 1 500 kg/ha. The relative abundances of antagonistic bacteria (Sphingomonas, Pseudomonas, Gemmatimonas, unidentified Gemmatimonadaceae, Lysobacter and Burkholderia), degrading bacteria (Ramlibacter), functional bacteria (Candidatus Solibacter, Bryobacter and Rhizomicrobium), rhizosphere probiotic bacteria (Flavisolibacter) at genus level were also increased after biofumigation. Biofumigation with marigold stalk showed a potential role in alleviating the obstacle of continuous tobacco plantation.