Lichens are composite organisms that produce a wide variety of secondary metabolites; many of the compounds have a high potential as bioactive compounds. The major limitations of using bioactive compounds from lichens is their slow growth rate and the damage to environmental populations caused by massive collection. The alternative to the massive collection of lichens in the field is their culture under laboratory conditions. We chose two related lichen species of Parmeliaceae that produce similar metabolites and isolated from spores in cultures placed under axenic conditions for over 550 days. From these cultures, we sampled 35 mg of each species from different culture media at two sampling times. The samples were analyzed using high-performance liquid chromatography (HPLC) to detect and identify major compounds. We found no differences in the metabolites produced within the species in comparisons between different culture media. Our results show that the mycobiont cultures produced different secondary metabolites than those found in natural lichen thalli. Moreover, different secondary metabolites between species and different metabolites over time were observed. We conclude that mycobiont cultures are a promising alternative for determining bioactive compounds and enhancing the efficiency of growth and production. These could be a good option for eco-friendly metabolite production.