Sterile cultured isolates of lichen-forming aposymbionts have not yet been used to investigate lichen-rock interactions under controlled conditions. In this study mycobionts and photobiont of the endolithic lichens Bagliettoa baldensis and Bagliettoa marmorea were isolated and inoculated with coupons of one limestone and four marbles commonly employed in the Cultural Heritage framework. After one year of incubation, microscopic observations of polished cross-sections were performed to verify if the typical colonization patterns observed in the field may be reproduced in vitro and to evaluate the receptivity of the five lithotypes to endolithic lichens. The mycobionts of the two species developed both on the surface of and within all the lithotypes, showing different penetration pathways which depend on mineralogical and structural features and highlight different receptivity. By contrast, algae inoculated with the coupons did not penetrate them. Observations suggest that the hyphal penetration along intrinsic discontinuities of rocks is a relatively fast phenomenon when these organisms are generally considered as slow-growing. Samples from limestone outcrops and abandoned marble quarries, colonized by the same species or other representatives of Verrucariaceae, showed penetration pathways intriguingly similar to those reproduced in vitro and highlighted that lichen-driven erosion processes only increase the availability of hyphal passageways after a long-term colonization. These results show that in vitro incubation of sterile cultured lichen-forming ascomycetes with rock coupons is a practicable experimental system to investigate the lichen-rock interactions under controlled conditions and, together with analysis in situ, may support decisions on conservative treatments of historical and cultural significant stone substrata.