This report describes the sequence of fungal colonization and the influence of biocide incorporation on paint films, determined using quantitative methods. Two buildings were painted with an acrylic paint, with and without an experimental biocide formulation containing a carbamate (carbendazin), N-octyl-2H-isothiazolin-3-one and N-(3,4-dichlorophenyl)N,N-dimethyl urea (total biocide concentration 0.25% w/w). One week after painting, the major groups of organisms detected were yeasts and Cladosporium. The yeast population fell to undetectable levels after the third week and this microbial group was not detected again until the 31st week, after which they increased to high levels on the 42nd week. Aureobasidium showed a pattern similar to the yeasts. The main fungal genera detected over the 42-week period were Alternaria, Curvularia, Epicoccum, Helminthosporium, Coelomycetes (mainly Pestalotia/Pestalotiopsis), Monascus, Nigrospora, Aureobasidium and Cladosporium. The latter was the main fungal genus detected at all times. The physiological factors controlling colonization are discussed. Cladosporium, Aureobasidium, Tripospermum and yeasts on the painted surfaces were all able to grow on mineral salts agar containing 10% sodium chloride. This is the first time that the genus Tripospermum has been reported on painted buildings. The fungal population on biocide-containing surfaces was significantly lower than on non-biocide-containing paint after 13 weeks and continued so to 42 weeks after painting, but there was no statistically significant difference in the level of fungal biodiversity.