This study aimed to explore the use of antioxidant enzyme activities (AEA) and biofilm metal accumulation capacity in natural communities as effect-based indicator of metal exposure in fluvial systems. To achieve these objectives, an active biomonitoring using fluvial biofilm communities was performed during 5 weeks. Biofilm was colonized over artificial substrata in a non-polluted site. After 5 weeks, biofilms were translocated to four different sites with different metal pollution in the same stream. The evolution of environmental parameters as well as biofilm responses was analysed over time. Physicochemical parameters were different between sampling times as well as between the most polluted site and the less polluted ones, mainly due to Zn pollution. In contrast, AEA and metal accumulation in biofilms allowed us to discriminate the high and moderate metal pollution sites from the rest. Zn, the metal with the highest contribution to potential toxicity, presented a fast and high accumulation capacity in biofilms. According to the multivariate analysis, AEA showed different responses. While catalase (CAT) and ascorbate peroxidase (APX) variability was mainly attributed to environmental stress (pH, temperature and phosphate concentration), glutathione-S-transferase (GST) changes were related to metal pollution. Glutathione reductase (GR) and superoxide dismutase (SOD) responses were related to both stress factors. AEA and metal accumulation are proposed as sensitive effect-based field methods, to evaluate biofilm responses after acute metal exposure (e.g. an accidental spill) due to their capacity to respond after few hours, but also in routinely monitoring due to their persistent changes after few weeks of exposure. These tools could improve the Common Implementation Strategy (CIS) of theWater Framework Directive (WFD) as expert group request.