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Surfactants are used to control microbial biofilms in industrial and medical settings. Their known toxicity on aquatic biota, and their longevity in the environment, has encouraged research on biodegradable alternatives such as rhamnolipids. While previous research has investigated the effects of biological surfactants on single species biofilms, there remains a lack of information regarding the effects of synthetic and biological surfactants in freshwater ecosystems. We conducted a mesocosm experiment to test how the surfactant sodium dodecyl sulfate (SDS) and the biological surfactant rhamnolipid altered community composition and metabolic activity of freshwater biofilms. Biofilms were cultured in the flumes using lake water from Lake Lunz in Austria, under high (300 ppm) and low (150 ppm) concentrations of either surfactant over a four-week period. Our results show that both surfactants significantly affected microbial diversity. Up to 36% of microbial operational taxonomic units were lost after surfactant exposure. Rhamnolipid exposure also increased the production of the extracellular enzymes, leucine aminopeptidase, and glucosidase, while SDS exposure reduced leucine aminopeptidase and glucosidase. This study demonstrates that exposure of freshwater biofilms to chemical and biological surfactants caused a reduction of microbial diversity and changes in biofilm metabolism, exemplified by shifts in extracellular enzyme activities. Key points: • Microbial biofilm diversity decreased significantly after surfactant exposure. • Exposure to either surfactant altered extracellular enzyme activity. • Overall metabolic activity was not altered, suggesting functional redundancy. [ABSTRACT FROM AUTHOR]