In this study we investigated the microbial contamination of 126 samples of photographic and cinematographic materials from 10 archival funds in the Czech Republic. Microorganisms were isolated from the light-sensitive layer by swabbing it with a polyurethane sponge. Microbial isolates were identified by MALDI-TOF MS (bacteria) or by phenotype testing and microscopy (fungi). Bacterial contamination was more abundant and more diverse than fungal contamination, and both were significantly associated with archives. The most frequently isolated fungal genera were Cladosporium, Eurotium, Penicillium, Aspergillus and Alternaria. The most frequently isolated bacteria were Gram-positive genera such as Staphylococcus, Micrococcus, Kocuria, Streptococcus and Bacillus. This bacterial and fungal diversity suggests that air is the main vehicle of contamination. We also analysed the impact of the type of material used for the carrier (paper, baryta paper, cellulose acetate and nitrate or glass) or the light-sensitive layer (albumen, gelatine, collodion and other) on the level and diversity of microbial contamination. Carriers such as polyester and cellulose nitrate may have a negative impact on bacterial contamination, while paper and baryta paper may have a partially positive impact on both fungal and bacterial contamination.