Historically valuable canvas paintings are often exposed to conditions enabling microbial deterioration. Painting materials, mainly of organic origin, in combination with high humidity and other environmental conditions, favor microbial metabolism and growth. These preconditions are often present during exhibitions or storage in old buildings, such as churches and castles, and also in museum storage depositories. The accumulated dust serves as an inoculum for both indoor and outdoor fungi. In our study, we present the results on cultivable fungi isolated from 24 canvas paintings, mainly exhibited in Slovenian sacral buildings, dating from the 16 to 21 centuries. Fungi were isolated from the front and back of damaged and undamaged surfaces of the paintings using culture media with high- and low-water activity. A total of 465 isolates were identified using current taxonomic DNA markers and assigned to 37 genera and 98 species. The most abundant genus was , represented by 32 species, of which 9 xerophilic species are for the first time mentioned in contaminated paintings. In addition to the most abundant xerophilic , , , and , xerophilic and , xerotolerant , , , and xerotolerant species were most frequent. When machine learning methods were used to predict the relationship between fungal contamination, damage to the painting, and the type of material present, proteins were identified as one of the most important factors and cracked paint was identified as a hotspot for fungal growth. species colonize paintings regardless of materials, while spp. can be associated with animal fat. Culture media with low-water activity are suggested in such inventories to isolate and obtain an overview of fungi that are actively contaminating paintings stored indoors at low relative humidity.