This study assesses the performance of several parameters as indicators of stream functional health in the context of the “Reference condition approach”. In 25 reference and 14 disturbed sites, from three catchments in Portugal (≈ 11200 km2), we measured sediments respiration rates; biofilms growth rates (AFDM m-2 d-1), chlorophyll a concentration (Chl a) and the Autotrophic index (AI) of biofilms in artificial substrates; biofilms biomass in natural substrates; and fungi biomass in incubated oak leaves; and total and microbial decomposition rates of Alnus glutinosa and Quercus robur leaves. At all sites, macroinvertebrates were sampled and 29 environmental variables were measured. We concluded that: decomposition of leaf litter was sensitive to disturbances and predictive models could be used to categorize the increasing levels of degradation; sediments respiration rates were successful in detecting impairment and were less variable across different types of streams than other parameters; biofilms growth in plastic artificial substrates may be a promising technique due to its easy and costless application and the ability to distinguish between disturbed and reference sites in terms of AFDM m-2 d-1, Chl a and AI. We concluded that functional parameters could be valuable complements of other indicators of stream health.