•11 fungal isolates from cultural heritage institutions’ interiors were screened for xerophilic trait.•Specially designed incubators were constructed to hold specific RH levels of 55%, 63% and 74.%•Fungal growth was monitored at low relative humidity on wood and glass supports painted by traditional artists’ paints.•Effects of pigments, support materials and strain variability are discussed. Even though contamination of painted artwork by xerophilic moulds frequently causes aesthetical, physical and/or biochemical biodeterioration, mould growth on paints, prepared from assorted traditional artists’ pigments, has yet to be systematically evaluated especially with regard to low relative humidity (RH) levels and painted support materials. Therefore, we investigated 11 fungal strains isolated mostly from cultural heritage institutions’ interiors for their potential to grow on egg tempera paint films prepared with different colouring agents and applied on wooden and glass supports which were maintained in monoculture in specially designed incubators at three different RH levels of 55%, 63% and 74%. The growth rate of mould over the surface was assessed using fluorescent microscopy after Calcofluor White staining. Additionally, these stains were screened for their xerophilic and hydrolytic potential using standard microbiological assays. Results show that when comparing growth rates on egg tempera paint films, 6 isolates grew exclusively on wood, exemplifying the greater susceptibility of this supporting material to mould attack. Prussian blue paint also stimulated the growth of 6 isolates, and the maximum overall expansion (38%) was observed on Prussian blue painted wood. RH was the key factor limiting growth, and at RH of 55% only a slight growth of 2 isolates was observed on Prussian blue painted wood. On the same samples incubated at RH of 63%, 10 isolates exhibited a moderate to strong growth and 4 of these showed an additional increase in growth at 74% RH. Paints consisting of artists’ pigments carmine lake or lead white in general completely prevented the development of moulds. Nevertheless, tolerance was species/strain dependant and the growths of isolates Cladosporium halotolerans EXF-15,333, Aspergillus niger EXF-14,897 and Aspergillus creber EXF-15,148 on lead white paint (containing ions and salts of heavy metal lead (Pb+2)) even exceeds 11%. Standard microbiological tests showed that all stains had hydrolytic potential and proved positive for xerophilic trait, nevertheless their ability to develop on egg tempera paint films was mostly dependant of very specific conditions.