Major part of the cultural heritage consists of objects of organic, plant material, canvas. As such, they are highly susceptible to fungal contamination, which by its action accelerate the deterioration of the canvas. One of the most suitable methods to stop the destructive action of fungi is gamma radiation. This paper investigates the resistance of linen mycobiota to gamma radiation as well as the resistance of the inoculated secondary colonizer type Cladosporium sphaerospermum. The samples were treated with a source at the Laboratory for radiation chemistry and dosimetry of Ruđer Bošković Institute. Absorption doses of 2, 7, 20 and 50 kGy were applied at two radiation rates of 0,1 and 8,6 Gy/s. Microbiological analysis of irradiated samples was carried out immediately after radiation and on the 14th, 28th and 56th day after radiation. Natural mycobiota of linen material includes species from the genera Penicillium, Cladosporium, Fusarium and yeast. The antifungal effect of gamma radiation is dependent on dose and dose rate. Fusarium spp., orange yeasts and Cladosporium spp. proved to be the most resistant to gamma radiation. The test results also set D10 value of 13,72 kGy for higher radiation rate (8,6 Gy/s). A chemical analysis of the linen material should be carried out in order to obtain better insight into the impact of certain doses and the radiation rates on the composition of the canvas. An interdisciplinary approach is required for the effective application of gamma radiation in the conservation of canvas objects of cultural heritage.