Biochar (BC) addition is widely used in agriculture to condition soils. However, the effects of BC addition on soil microbial community diversity and composition in karstic regions are unclear, especially after long-term application. To address this knowledge-gap, a field experiment was established to examine changes in soil physicochemical properties and microbial communities following six years of BC amendment. BC was applied to calcareous soils in a karstic region of southwestern China at four levels (w/w): 0%, 1.0%, 5.0%, and 10%. Bacterial community composition was then investigated in both the rhizosphere and bulk soils by 16S rRNA gene sequencing on the Illumina MiSeq Platform. BC addition increased soil pH, total carbon (TC), total nitrogen (TN), and total hydrogen (TH) contents in the rhizosphere and bulk soils. In addition, BC amendment was associated with changes in soil bacterial community compositions and diversities, especially at higher BC application levels. The relative abundances of Gemmatimonadetes increased in rhizosphere soils with increasing BC amendment, while those of the Bacteroidetes, Firmicutes, and Cyanobacteria decreased. The relative abundances of Proteobacteria and Chloroflexi increased in bulk soils with increasing BC application levels, while those of the Bacteroidetes and Verrucomicrobia decreased. Canonical correspondence analysis indicated that bacterial community composition was related to soil characteristics including pH, TC, TN, and TH contents in both rhizosphere and bulk soils. Importantly, variations in these soil parameters were closely associated with BC application rates. These results indicate that long-term BC application significantly impacts soil bacterial community characteristics in karstic regions via modulation of soil physiochemical properties.