Over time, cultural heritage has become a key for comprehending and developing our society at an individual and group level, as it provides fundamental information about our origins, specific temporary space, materials' availability using current technology, artist's intention, and site weather conditions. Here, we investigated the physical characteristics of an Italian large-format artwork diptych, located on the ceiling of the National Theater of Costa Rica, to evaluate its pictorial palette as well as the tropical climatological conditions and the fungal aerial spore concentration. We characterized the role of two innovative software tools, as they have direct connection with an effective microbiological sampling and description of secrets encompassed in each pictorial layer during the process of creation and intention. We further found that eight genera of calcareous nannofossils could be associated with the optical properties of the artwork and the effects that the artist wanted to portray through his creative process.