Land-use change from natural to managed agricultural ecosystems significantly impacts soil bacterial diversity and function. The Eastern Free State (EFS) is one of the most productive agricultural regions in South Africa. However, no studies aiming to understand the changes in bacterial diversity, composition and function due to land-use change in this area have been conducted. This study investigated, using high-throughput 16S rRNA gene amplicon sequencing, the effects of long-term agriculture on bacterial diversity, composition and putative function in the EFS by comparing microbiomes from lands that have been under agronomic activity for over 50 years to those from uncultivated land. Results indicate that agriculture increased bacterial diversity. Soil chemical analysis showed that land-use shifted soils from being oligotrophic to copiotrophic, which changed bacterial communities from being Actinobacteria dominated to Proteobacteria dominated. Predictive functional analysis using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) suggested that agricultural soil was abundant in genes associated with plant fitness and plant growth promotion, while non-agricultural soil was abundant in genes related to organic matter degradation. Together, these results suggest that edaphic factors induced by long-term agriculture resulted in shifts in bacterial diversity and putative function in the EFS.