Crop rotational diversity can improve crop productivity and soil health, and boost soil microbial diversity. This research hypothesized that a 3-year rotation of corn-soybean-wheat (CSW), compared with a 2-year corn-soybean (CS) rotation, would result in a more diverse and more complex soil bacterial community, together with a greater abundance of beneficial bacteria. This was evaluated in a replicated experiment established in 2013 at two locations in Ohio (United States). The soil bacterial communities under soybean were compared between CS and CSW at both studied sites in 2018 and 2019, through 16S ribosomal DNA amplicon metabarcoding. Experimental site was the main driver of bacterial richness and evenness. Significant effects on bacterial community composition were observed in response to the interaction between site, rotational sequence, and year of study. Eight bacterial amplicon sequence variants were identified within all CSW treatments and were not present in CS. Several taxa were differentially abundant between rotation treatments, including the genera Ralstonia being more abundant in CS. Co-occurrence networks, including hub taxa, were generally different between rotation treatments and year, with more structure observed in CSW networks for one of the studied sites. Few bacterial genera were consistently identified as hubs across all networks, including an unidentified member of order Acidobacteriales, while other hubs were unique for CSW networks, including members of the family Gemmatimonadaceae. Finally, the composition of the bacterial communities at the northwestern site positively correlated with plant biomass and active carbon, whereas more recalcitrant pools (total carbon and organic water) correlated with the bacterial communities at the western site.