Numerous studies have reported the dynamics of microbes when biochar was applied, whereas the information on the alterations of bacterial community after application of rapeseed straw-derived biochar is limited. A pot experiment with two rapeseed straw-derived biochar application treatments (with biochar application at the rate of 200 g/pot, C1, and without biochar application, 0 g/pot, C0) was conducted. No significant differences were observed in the number of operational taxonomic units, observed species, Shannon index, Simpson index, Chao1, ACE, and phylogenetic diversity whole tree between the C1 and C0 treatments. Taxonomic analysis at the phylum level showed that the abundances of Bacteroidetes and Parcubacteria were higher in the C1 treatment compared to the C0 treatment, while Acidobacteria, Chloroflexi, Rokubacteria, Berkelbacteria, and Latescibacteria were observed with higher abundance in the C0 treatment compared to the C1 treatment. Taxonomic analysis at the genus level showed that the abundances of Gracilibacter, Lentimicrobium, unidentified Rikenellaceae, Hydrogenophaga, and Bacillus were higher in the C1 treatment compared to the C0 treatment, while Candidatus Solibacter, Candidatus Koribacter, and Lutispora abundances were found to be higher in the C0 treatment compared to the C1 treatment. Obvious clusters were observed between the C1 and C0 treatments in both principal component analysis and nonmetric multidimensional scaling. These results indicate that soil bacterial community was altered after rapeseed straw-derived biochar was applied.