Jacaranda mimosifolia(Bignoniaceae) is an important economic, ecological and ornamental tree native to South America. It is cultured in squares, parks and gardens throughout America, Europe, South Africa and Asia. During plant-microbe interactions, plant growth promoting bacteria (PGPB) could optimize plant production by mitigating undesirable features which trouble ex vitro transferring. So, the aim of this work was to evaluate the effect of biofertilization with A. brasilense Az39 (as reference strain), Methylobacterium sp. L10 and Stenotrophomonas sp. L20 on micropropagation of Jacaranda mimosifolia. Woody Plant Medium (WPM) was used for seeds germination whereas Murashige and Skoog salts with Gamborg vitamins (MSG) supplemented with 6-benzylaminopurine (BA) were used in multiplication stage and supplemented with indole butyric acid (IBA) were used in rooting stage. All stages were inoculated or not with Az39, L10 or L20. Culture system, bacteria inoculation in each micropropagation stage and their interaction were significant. Methylobacterium sp. L10 and Stenotrophomonas sp. L20 decreased fungal contamination at initial establishment by seeds. In addition, Methylobacterium sp. L10 inoculation increased the multiplication rate by 94% relative to non-inoculated shoots. Stenotrophomonas sp. L20 inoculation in combination with a pulse with 30 μM IBA increased in vitro and ex vitro rooting percentage. In the acclimatization stage, 95% of ex vitro-rooted plants inoculated with Stenotrophomonassp. L20 survived. The strains used at this work could act as biological control agents and could be used for biofertilizers development.