The maintenance of forest formations under a scenario of climatic changes and unsustainable anthropic use of natural resources is vital for the future of mankind. In this context, there is a rising agreement that underutilized crops may be important for the future production of food, as they tend to be more adapted to stressful conditions than domesticated crops. Eugenia uni ora L. (Myrtaceae) is a key species in different forest formations with ecological, economical, and medicinal importance. The present work aimed at establishing an in vitro culture procedure for the mass micropropagation of E. uni ora genotypes through the organogenesis route. Nodal segments from in vitro germinated seeds 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 protocol provided in this study may be helpful for the mass production of seedlings for degraded areas recovery or commercial orchard implementation, as well as in genetic improvement programs for E. uni ora.