Major part of cultural heritage is made up of paper-based materials. As such, they are highly susceptible to fungal contamination, which causes paper to decay. One of the most convenient methods of stopping the destructive action of fungi is gamma irradiation. This paper was aimed to assess the influence of gamma irradiation on the naturally occurring mycobiota as well as artificially inoculated secondary colonizer Cladosporium sphaerospermum. Inoculated samples have been treated by the Laboratory for radiation chemistry and dosimetry of Ruđer Bošković Institute source. Doses of 2, 7, 20 and 50 kGy were applied at two dose rates of 0,1 and 8,6 Gy/s. Microbiological analysis of irradiated samples was conducted immediately after radiation and on 14, 28 and 56 days after radiation. Fungicidal effect of gamma radiation was dose and dose rate-dependent. From the obtained results it is apparent that the lowest dose of 2 kGy is not effective in reducing mold at either lower or higher dose rates. While higher doses of 7, 20 and 50 kGy at higher dose rate effectively reduced the naturally occurring mycobiota as well as artificially inoculated secondary colonizer C. sphaerospermum. This is also confirmed by the calculated value D10 (decimal reduction dose), the dose required to inactivate 90% of the initial number of molds in the sample, which is 11.29 kGy for C. sphaerospermum and the rate of radiation 0.1 Gy/s, and for 8.6 kGy is 4.35 kGy. Thus, it is necessary to find an appropriate dose which will reduce the microorganisms to an acceptable level while at the same time will have a minimal negative effect on the chemical composition of the paper, so further testing in this area is required. To conclude, for the efficient use of gamma irradiation in conservation of paper-based cultural heritage objects, an interdisciplinary approach is needed.