Beneficial soil microorganisms, particularly plant probiotic bacteria (PPB), play a pivotal role in promoting plant growth, development, and overall health through root colonization. PPB-based biofertilizers offer a sustainable and eco-friendly alternative to conventional agricultural inputs. This study evaluates the plant probiotic potential of three native bacterial strains Rhizobium sp. ACO-34A, Sinorhizobium mexicanum ITTG R7^T, and Sinorhizobium chiapasense ITTG S70^T to enhance the growth, quality, and sugar content of Agave tequilana. A comprehensive genomic and functional analysis was conducted for each strain to assess their plant probiotic traits. Additionally, a greenhouse inoculation assay was performed on six-month-old agave seedlings at the “piña” stage to evaluate the effects of these strains on plant growth and sugar content. Comparative genomic analysis revealed that these rhizobial strains harbor genes associated with key plant probiotic traits, reinforcing their role in enhancing plant development. The results demonstrated significant effects (p < 0.05) on growth and sugar content in inoculated plants. ACO-34A increased plant height by 35.4%, fresh weight by 41.5%, and inulin content by 57.3%, while ITTG-R7^T showed improvements of 26.4%, 35.2%, and 38.2%, respectively, compared to the control, and ITTG S70^T also exhibited enhancements, although to a lesser extent, with increases of 23.5% in plant height, 28.9% in fresh weight, and 31.2% in inulin content. These findings highlight the biofertilizer potential of these native rhizobial strains, particularly Rhizobium sp. ACO-34A, positioning them as promising candidates for the sustainable cultivation of A. tequilana.