Peanut (Arachis hypogaea L.) forms root nodules that host microbial communities influencing plant nutrition and stress tolerance, and herbicide use may act as an environmental filter altering the cultivable nodule microbiota. This study isolated and characterized bacteria from peanut nodules collected in fields with and without imazapic application in Jaboticabal, São Paulo, Brazil. Eight isolates were obtained, and one hemolytic strain was excluded after pathogenicity screening. Based on 16S rRNA gene sequencing and phylogenetic analysis, the isolates were identified as Bacillus aerophilus, Bacillus inaquosorum, Bacillus subtilis, Bradyrhizobium yuanmingense, Burkholderia lata, and Rhizobium tropici. Nodules from herbicide-treated plants yielded exclusively Bacillus spp., whereas those from non-treated plants showed greater taxonomic diversity. Molecular screening detected genes associated with biological nitrogen fixation (nifH) and nodulation (nodA, nodB, nodC, nodD), indicating potential functional capacity. In greenhouse assays, the isolates showed strain-dependent effects on early plant development, with pronounced responses in root growth and nodulation. Burkholderia lata and bacterial consortia enhanced root development and nodulation, with performance comparable to the commercial inoculant SEMIA 6144. Herbicide management shapes the cultivable nodule microbiota, and selected isolates show potential as bioinoculants for peanut production systems.