The role of fungi in the biodeterioration of frescoes has been investigated by means of transparent adhesive tape and by swabbing the surface of selected areas as well as by a study of the materials used in restoration. In the Ognissanti church at Florence a large fungal population was found on two 15th century frescoes, one by Botticelli of ‘St Augustine’ and the other by Ghirlandaio of ‘St Jerome’. Twenty-three species were identified, 15 on the Botticelli where the majority of the colony forming units (cfu) were Penicillium brevi-compactum Dierckx (61%) and P. purpurogenum Stoll (18%), and 13 on the Ghirlandaio where Aspergillus versicolor (Vuill.) Tiraboschi was the most common (74%), together with Cladosporium sphaerospermum Penz. (22%). The materials often used in restoration namely calcium caseinate, masonite and animal glue were tested and found to be suitable substrates for species of Aspergillus and Penicillium but not for Cladosporium cladosporioides and C. sphaerospermum.