Orchids are valued as ornamental plants, bioindicators, and medicinal plants, which implies that some species may be over-collected. Some inhabit very fragile environments and are under threat by the misuse of habitats and anthropogenic impacts. The search for beautiful plants and flowers has increased the number of facilities for micropropagation either by seeding or by cloning plants using in vitro techniques. However, not all species have appropriate media for growth and development that would help in conservation efforts. Cyrtopodium aliciae is an endemic species of rupestrian grassland in Brazil t. It has appeal as an ornamental plant or for use in hybridisation programs dueo its small size and white brownish-purple dotted flowers. This study compared three different media, namely ½ concentration Murashige and Skoog (MS), Vacin and Wendt, and Knudson C, during plant growth and their effect on the acclimatization of Cyrtopodium aliciae. The number and length of shoots and roots, increase in mass, and survival in vitro and ex vitro were analyzed. The experiment was conducted as completely random with a factorial arrangement of treatments (3 × 3) with 10 repetitions per treatment containing 10 plants for the in vitro experiment and 3 repetitions of 10 plants for the ex vitro experiment. Cyrtopodium aliciae performed better in the ½ concentration MS medium with a higher increase in mass, plant development, and survival under both in vitro and ex vitro conditions.