Biochar has been shown to affect soil microbial diversity and abundance. Soil microbes play a key role in soil nutrient cycling, but there is still a dearth of knowledge on the responses of soil microbes to biochar amendments, particularly for longer-term or repeated applications. We sampled soil from a field trial to determine the individual and combined effects of newly applied (1 year ago), re-applied (1 year ago into aged biochar) and aged (9 years ago) biochar amendments on soil bacterial communities, with the aim of identifying the potential underlying mechanisms or consequences of these effects. Soil bacterial diversity and community composition were analysed by sequencing of 16S rRNA using a Miseq platform. This investigation showed that biochar in soil after 1 year significantly increased bacterial diversity and the relative abundance of nitrifiers and bacteria consuming pyrogenic carbon (C). We also found that the reapplication of biochar had no significant effects on soil bacterial communities. Mantel correlation between bacterial diversity and soil chemical properties for four treatments showed that the changes in soil microbial community composition were well explained by soil pH, electrical conductivity (EC), extractable organic C and total extractable nitrogen (N). These results suggested that the effects of biochar amendment on soil bacterial communities were highly time-dependent. Our study highlighted the acclimation of soil bacteria on receiving repeated biochar amendment, leading to similar bacterial diversity and community structure among 9-years old applied biochar, repeated biochar treatments and control.