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Background Eugenia uniflora L (Myrtaceae) is a native shrub from Brazil and is used in traditional medicine and also in the food industry to produce different types of juices, jellies, ice cream, and can also be consumed unprocessed. This species is propagated by seeds, which have irregular germination leading to low producer orchards affecting its economic viability for large‐scale production and the quality of seedlings. Thus, the main goal of this study was to evaluate different in vitro germination conditions and the effects of substrate and fertilizer on ex vitro plantlets development. Results Seeds germinated in MS0 under light had the highest speed germination index (1.63). The highest root system length was observed in seeds cultivated in MS + 1 μmol GA3 in the presence of light (12.85 cm). Plantlets developed under MS + 1 μmol GA3 in the presence of light, showed the highest dry weight mass (29.7 mg). The three‐way ANOVA indicated that the type of fertilizer, culture medium, and light conditions separately showed significant effects on shoot length (p < 0.0001, 0.0278, and 0.036 respectively), root collar diameter (p < 0.0001, 0.0428, and 0.0165, respectively) and also for the fresh and dry weight of the shoot, just by the type of fertilizer (p < 0.0001) and culture medium (p < 0.05) used. Conclusion It was concluded that MS0 under light is the most suitable condition for Brazilian cherry germination and commercial substrate plus the controlled release NPK 15‐9‐12 fertilizer provides better plant growth.