Mastic, one of the best natural varnishes, is frequently used as protective and finishing layer or as component of oleo-resinous media in paintings, both in the past and currently. However, this resin is affected by complex deterioration processes which can change its characteristics and thus the visual aspect of works of art. The alteration processes caused by radiation have been widely studied, but there is a lack of information on the biodeterioration of this natural product. In this paper, fungi from collections as well as from oil paintings of the Fine Arts Museum of Granada (Spain) were inoculated onto slides covered with mastic. The samples, after an incubation period of 15 days, were analysed by gas chromatography-mass spectrometry (GC-MS) to identify the chemical changes undergone, and a visual monitoring of the samples was performed to determine the formation of mycelia onto solidified resins. Major changes were detected in Chrysonilia sitophila, Phoma herbarum, and P. chrysogenum, showing evidence of alteration processes caused or favoured by these microorgamisms.