An experiment was conducted with tobacco (Nicotiana tabacum L.) grown in a Cd- and Pb-contaminated calcareous soil amended with 0.0, 1.0, 2.5, and 5.0% (w/w) tobacco stalk biochar (BC). The BC amendment significantly increased organic matter, total C, N, P, and K contents of soil, and the C/N ratio. Bioavailable metal concentrations (DTPA extraction) decreased by increasing BC application rate. The 5.0% BC amendment significantly decreased the DTPA-extractable Cd and Pb by 10.4 and 13.6%, respectively. Correspondingly, the bioaccumulation and translocation factors of Cd and Pb also decreased by increasing the BC addition rates and this indicated that BC inhibited the uptake and transfer of both Cd and Pb by tobacco plants. Moreover, high-throughput sequencing revealed that BC increased Chao1 richness, Shannon's diversity and Simpson's diversity of bacterial communities of soil. The relative abundance and genera composition of Adhaeribacter, Rhodoplanes, Pseudoxanthomonas, and Candidatus Xiphinematobacter increased under BC treatments, while those of Kaistobacter, Lacibacter, and Pirellula decreased. Overall, BC increased soil nutrients (C, N, P, and K contents), enhanced bacterial diversity indexes and richness, and changed the bacterial community composition, which may all have contributed to reduce the mobility and bioavailability of both Cd and Pb in a calcareous soil.