Trifolium rubens L. is a leguminous plant “Preferential Attention”, according to the Catalog of Protected Flora of Castile and Leon (Spain). In this study we aimed to analyze the potential of three bacterial strains of the genus Rhizobium to improve the growth and development of this plant. All three strains produced 3-indoleacetic acid (IAA), but the strain ATCC 14480 produced the most. In addition, all strains produced biofilms and cellulases, although in different quantities. The synthesis of these products has been described as being related to the processes of the adherence of bacteria to the plant root surface and their entrance into the plant, respectively. In addition, in vitro assays and assays conducted under controlled and sterile conditions were performed, showing that the three strains were capable of nodulating T. rubens L. and effectively fixed nitrogen for the plant. These results were corroborated by morphological and histological analysis of nodules. Finally, greenhouse assays determined the effects of the strains under more competitive conditions, and it was concluded that inoculated plants presented greater lengths and weights, both aerial and radicular, and also chlorophyll and nitrogen content compared to the uninoculated controls.