Background: Due to their huge biodiversity and the capability to produce a wide range of secondary metabolites, lichens have a great potential in biotechnological applications. They have, however, hardly been used as cell factories to date, as it is considered to be difcult and laborious to cultivate lichen partners in pure or co-culture in the laboratory. The various methods used to isolate lichen fungi, based on either the ascospores, the conidia, or the thallus, have so far not been compared or critically examined. Therefore, here we systematically investigate and compare the known methods and two new methods to identify the most suitable technology for isolation of fungi from lichens. Results: Within this study six lichen fungi species were isolated and propagated as pure cultures. All of them formed colonies within one month. In case of lichens with ascocarps the spore discharge was the most suitable method. Spores were already discharged within 2 days and germinated within only four days and the contamination rate was low. Otherwise, the soredia and thallus method without homogenization, as described in this work, are also well suited to obtain pure fungal cultures. For the isolation of algae, we were also successful with the thallus method without homogenization. Conclusion: With the methods described here and the proposed strategic approach, we believe that a large proportion of the lichen fungi can be cultivated within a reasonable time and efort. Based on this, methods of controlled cultivation and co-cultivation must now be developed in order to use the potential of lichens with regard to their secondary metabolites, but also for other applications. Keywords: Ascomycota, Lichen cultivation, Isolation method, Colony development, Pure culture, Axenic culture, Co-culture, Secondary metabolites, Biotechnology.