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Fungi are important contaminants of historic canvas paintings worldwide. They can grow on both sides of the canvas and decompose various components of the paintings. They excrete pigments and acids that change the visual appearance of the paintings and weaken their structure, leading to flaking and cracking. With the aim of recognizing the most dangerous fungal species to the integrity and stability of paintings, we studied 55 recently isolated and identified strains from historic paintings or depositories, including 46 species from 16 genera. The fungi were categorized as xero/halotolerant or xero/halophilic based on their preference for solutes (glycerol or NaCl) that lower the water activity (aw) of the medium. Accordingly, the aw value of all further test media had to be adjusted to allow the growth of xero/halophilic species. The isolates were tested for growth at 15, 24 °C and 37 °C. The biodeterioration potential of the fungi was evaluated by screening their acidification properties, their ability to excrete pigments and their enzymatic activities, which were selected based on the available nutrients in paintings on canvas. A DNase test was performed to determine whether the selected fungi could utilize DNA of dead microbial cells that may be covering surfaces of the painting. The sequestration of Fe, which is made available through the production of siderophores, was also tested. The ability to degrade aromatic and aliphatic substrates was investigated to consider the potential degradation of synthetic restoration materials. Xerotolerant and moderately xerophilic species showed a broader spectrum of enzymatic activities than obligate xerophilic species: urease, β-glucosidase, and esterase predominated, while obligate xerophiles mostly exhibited β-glucosidase, DNase, and urease activity. Xerotolerant and moderately xerophilic species with the highest degradation potential belong to the genus Penicillium, while Aspergillus penicillioides and A. salinicola represent obligately xerophilic species with the most diverse degradation potential in low aw environments.