Microbial deterioration frequently affects photographic collections, and has been considered a major cause of gelatine deterioration. However, there are no detailed studies of this topic. The goal of this study was to isolate and to identify the microbial contaminants of gelatine emulsion photographs belonging to three Lisbon collections: Bourdin de Macedo collection from the city council archive, Horacio Novais collection from the Calouste Gulbenkian Foundation Art Archive, and the collection from the Portuguese Geographical Society. Several isolation methods were tested. In about 60 strains isolated from 131 samples, the most common fungi belong to the genera Penicillium (35%) and Aspergillus (23%), followed by the genus Cladosporium (12%). The microorganisms identified were discussed in relation to the photographic materials affected and the collections.