Eugenia uniflora L. (Myrtaceae) is a key species in different forest formations with ecological, economic, and medicinal importance. This study aimed at establishing an in vitro culture procedure for the micropropagation of E. uniflora genotypes through organogenesis. Nodal segments were introduced in a semi-solid medium for organogenesis establishment and posteriorly cultivated in immersion systems for shoot development and rooting. Different culture media, plant growth regulator concentrations, and immersion systems were tested. The highest rate of shoot generation was obtained with 2.0-2.5 mu M BAP + 0.5 mu M of NAA. The measures related to rooting did not differ among the tested immersion systems, but the biomass accumulation and leaf area were significantly higher in the twin-flasks system. There were no significant differences in chlorophyll contents among the immersion systems. A significantly higher content of carotenoids was observed in the permanent immersion system with gas exchange. The protocol provided in this study may be helpful for the mass production of seedlings for commercial orchard implementation, as well as in genetic improvement programs for E. uniflora.