Biological nitrogen fixation performed by diazotrophic bacteria is a vital process for agricultural and environmental sustainability. In recent years, bacterial classification has been based on genomic data, accelerating our understanding about the diversity, and resulting in the description of several new species. In this study, four strains (CNPSo 3140T, CNPSo 3235, CNPSo 3236 and CNPSo 3237) trapped by Phaseolus vulgaris and Mimosa pudica from soil samples of the Brazilian Atlantic Forest biome (Mata Atlântica) were submitted to polyphasic analysis to investigate their proper classification within the genus Mesorhizobium. The 16S rRNA gene phylogram showed that the strains present sequences identical to those of Mesorhizobium acaciaeand Mesorhizobium plurifarium, not allowing a clear taxonomic classification; however, when using multilocus sequence analysis methodology, the strains were grouped into a well-supported distinct clade, with <94.5 % nucleotide identity with the other species of the genus. The average nucleotide identity of CNPSo 3140T genome showed values below the threshold in relation to the closest species, of 89.75 % with Mesorhizobium plurifariumand of 88.83 % with Mesorhizobium hawassense; the digital DNA-DNA hybridization values were 39 and 37.70 % with the same species, respectively. Nodulation gene (nodC) phylogeny positioned the strains in an isolated cluster, showing greater similarity to Mesorhizobiumshonense. All data obtained in this study support the description of the novel species Mesorhizobiumatlanticum sp. nov. The type strain is CNPSo 3140T (=ABIP 206T=LMG 30305T=U1602T), isolated from a nodule of Phaseolus vulgaris.