Conservation of hypogea and their accessibility by the visitors is a hard question, due to the interaction of different factors such as the intrinsic characteristics of the hypogeal environments and the presence of public. A particular case is represented by the Mithraeum of Marino Laziale, located a few kilometers away from Rome and accidentally discovered in the 1960s. The uniqueness of the discovery was the presence of a well-preserved painting of the Mithraic scene (II century A.D.) probably due to the oblivion of the place of worship over the centuries as well as the isolation from the outdoor environment. Unfortunately, despite a recent complete restoration and recovery of the archaeological area, which ended in 2015, the area was never open to the visitors and only two years after completing the works it was no longer safe to use. Hence, the need for a new planning of interventions starting from the deep knowledge of this cultural heritage and from the analysis of past incorrect actions to arrive at the opening—without any risk for the archaeological findings and visitors—and management of this site, never exposed to the public. Therefore, since 2018 a diagnostic campaign and microclimate monitoring have been started. The data collected during the two years of investigations have been fundamental to assess the conservation state of the hypogeal environment and the potential risks for the preservation of the three paintings (the Mithraic scene and two dadophores). Long-term monitoring of indoor environmental conditions assumes the role of an essential tool for the planning of preventive conservation strategies but also for the control of the site after its opening to the visitors. Furthermore, the characterization of the microclimate is non-invasive, sufficiently economical and accurate. In this paper, the characterization of surfaces in the Mithraic gallery through optical microscopy, UV fluorescence/imaging techniques, FT-IR spectroscopy, XRD and the microclimatic parameters variation in the presence or absence of visitors are used to define the strategies for the opening and fruition of the Mithraeum. The strategies for the sustainable fruition of this unique archaeological site have been defined through a conservation protocol approved by the Italian Ministry of Cultural Heritage and necessary for the site managers and curators of the Municipality of Marino Laziale to finally support its opening.