Lisianthus (Eustoma grandiflorum (Raf) Shinn.) is an excellent ornamental cut variety because it has flowers with good vase life. Seed propagation is difficult due to the long production time (it takes up to 100 days for transplanting) and the small size of its seeds. In vitro germination may produce healthy and homogeneous seedlings and may be used as a model system to study the effect of plant growth promoting rhizobacteria. Azospirillum brasilense is widely studied for its ability to colonize and promote various plant species growth, especially by increasing root development and plant biomass. This work aimed to evaluate physiological, morphological and anatomical aspects involved in vitro germination of E. grandiflorum inoculated with Azospirillum brasilense Az39. Seeds of E. grandiflorum (F1 double big. Arena III Pink) were disinfected with 10% NaClO and culture in MS medium. Inoculation of A. brasilense Az39 was performed with 106 and 107 cfu on seed surface at sowing. Non-inoculated seeds were used as controls. Biofertilization generated earliness and higher germination percentages. Furthermore, bacterization with 106 cfu significantly increased number of leaves, leaf area, fresh and dry weight of seedlings, length and number of roots, diameter of the vascular bundle, leaves thickness and root thickness, among other parameters. These results suggested that inoculation of lisianthus with A. brasilense Az39 could improve seedlings and lead to a shorter time production.