Aquatic microfungi play a fundamental role in organic matter decomposition in fluvial ecosystems. These micro-organisms degrade leaf recalcitrant compounds like lignin, thereby enhancing the utilization of organic material by the microbial community. The main input of allochthonous organic matter in Mediterranean streams occurs during the autumn. In-stream breakdown processes can be affected by high physical abrasion during flooding but changes in stream water chemistry may also affect decomposition enzymatic activities of stream microorganisms. We measured two ligninolytic activities (phenol oxidase and peroxidase) and a cellulolytic activity (cellobiohydrolase) in leaves, branches, sand and gravel substrata in a reach of a Mediterranean stream. Highest ligninolytic activities were measured in biofilm developed on inorganic substrata (sand and gravel) where also accumulated the highest fungal biomass (ranging from 3.3 x 10-4 to 7.22 mg Ergosterol gAFDM-1) especially in sand substrata. Conversely, cellulolytic activities were significantly higher in biofilm on organic substrata (leaves and branches). Physical and chemical factors, such as discharge and stream water nutrient concentration (Dissolved Inorganic Nitrogen) were affecting enzymatic activities, particularly enhancing the phenol oxidase. Moreover, the chemical composition of the available OM (high cellulose in leaves, high lignin in detritic material) strongly influenced the decomposition activity in each biofilm. A precise description and quantification of the benthic substrata was used to obtain enzymatic activity values in terms of stream reach. Those results showed a temporal pattern in the decomposition activities in the reach, beginning with the decomposition of cellulose (October) followed by lignin compounds (November and December)