Filamentous fungi have been constantly recovered from diverse aquatic environments including drinking water distribution systems. Although most of the works are focused on the study of planktonic form, recent researches have shown that fungi develop biofilm within these systems. In this study, Aspergillus sp. (section Nigri), Aspergillus sp. (section Flavi), Alternaria sp., Botrytis sp., Cladosporium sp., and Penicillium sp. recovered from water biofilms were used to evaluate their capability to grow as biofilms under laboratorial conditions. Morphological and physiological characteristics were analysed using image analysis and biomass and cell activity estimation. All six isolates were able to form biofilm, though different patterns of development were observed. Only Alternaria sp. formed biofilm in water over 24？h of analysis. MEB was shown to be the best culture media for biofilm formation. A direct correlation between biomass and cell activity was not observed, but biomass values and morphological parameters, that is, monolayer and EPS production, were directly correlated. Thus, the results present here highlight the capability of fungi to form biofilms and the emergent necessity to standardize methods for further research in this area. 1. Introduction Filamentous fungi (ff) have been frequently isolated from aquatic environments such as rivers, streams, lakes, and sea [1]. Water distribution systems (WDS) are nowadays seen as complex aquatic environments in which high diverse microorganisms cohabit, including fungi [2]. Regardless of their importance for human health, little is known about microbial ecology of ff within WDS. Moreover, the focuses of microbial water quality studies still remain on monitoring planktonic microorganisms, despite scientists’ awareness that the majority of microorganisms live together as biofilms [3]. Due to its important role in the environment, industry, and medicine, the understanding of mechanisms of biofilm formation has become the focus of biofilm research. Research in ff biofilm in WDS has only recently received attention [4–6]. Most of previous mycological studies are focused on pathogenic fungi, for example, Candida spp. [7–9], Aspergillus fumigatus [10–12], and opportunistic zygomycetes [13]. Increased resistance against antimicrobials is a well-known and still worrying clinical relevant biofilm feature, and wherefore these studies have especially established suitable methods for antimicrobial biofilm susceptibility assay [14–16]. Ramage et al. [17] reported the importance of fungal biofilm phenotype concept in
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