The use of the GreenTray® TIS bioreactor for the in vitro analysis of the interaction between plantlets and two Plant Growth-Promoting Microorganisms (PGPMs) (Pseudomonas oryzihabitans PGP01 and Cladosporium ramotenellum PGP02) is reported herein. This in vitro culture system improved micropropagation of Prunus Rootpac 20® rootstock, showing greater shoot length and fresh weight compared with culture in semisolid agar-containing medium. Plant responses in co-culture with the PGPMs, their ability to control endophytes growth in the culture media and hormonal changes associated to plant growth were studied in the GreenTray® culture system. Inoculation with P. oryzihabitans PGP01 had no significant effects, whereas C. ramotenellum PGP02 considerably reduced endophytes population after 5 days post inoculation. However, the fungi overgrew the plants roots seriously reducing their growth. These results suggested the effect of the medium pH as a factor to control microbial endophytic growth in the culture medium. A higher P. oryzihabitans PGP01 concentration was inoculated in culture media previously adjusted to pH 5.7 or 7, observing an increased number of roots when plantlets were co-cultured for 15 days with the bacterium in medium adjusted to pH 5.7. This was associated with changes in the levels of IAA in the culture medium, and higher leaf chlorophyll contents than in the non-inoculated cultured plantlets. P. oryzihabitans PGP01 was not able to reduce endophytes growth in either of both media pHs. Altogether, the GreenTray® bioreactor was shown as an efficient system to in vitro micropropagate RP-20 explants as well as to monitor its interaction with PGPMs.