The most representative bacterium (Pseudonocardia sp.) and fungus (Fusarium sp.) from the microbial communities of a cave containing paleolithic paintings were isolated and their growth on natural substrates assessed. Growth was tested at the in situ and optimal, laboratory growth temperature. Development was analyzed with and without supplemented nutrients (glucose, ammonium, phosphate, peptone). Results showed that the assayed bacterium on natural substrate was able to develop best at in situ temperature and the addition of organic nutrients and/or phosphate enhanced its growth. The growth of the assayed fungus, however, was limited by low temperature and the availability of ammonium. These results confirm a differential behavior of microorganisms between the laboratory and the natural environments and could explain previous invasion of fungi reported for some caves with prehistoric paintings.