Abstract Globally, freshwater ecosystems are warming at unprecedented rates and northern temperate lakes are simultaneously experiencing increased runoff of humic substances (brownification), with little known consequences for future conservation of biodiversity and ecosystem functioning. We employed an outdoor mesocosm experiment during spring and summer to investigate the combined effects of gradually increasing warming and brownification perturbations on the phytoplankton community structure (biodiversity and composition) and functioning (biomass). While we did not observe overall significant treatment effects on total phytoplankton biomasses, we show that predicted increases in warming and brownification can reduce biodiversity considerably, occasionally up to 90% of Shannon diversity estimates. Our results demonstrate that the loss of biodiversity is driven by the dominance of mixotrophic algae (Dinobryon and Cryptomonas), whereas several other phytoplankton taxa may be temporarily displaced from the community, including Cyclotella, Desmodesmus, Monoraphidium, Tetraedron, Nitzschia and Golenkinia. The observed loss of biodiversity coincided with an increase in bacterial production providing resources for potential mixotrophs along the gradient of warming and brownification. This coupling between bacterial production and mixotrophs was likely a major cause behind the competitive displacement of obligate phototrophs and supports evidence for the importance of consumer–prey dynamics in shaping environmental impacts on phytoplankton communities. We conclude that warming and brownification are likely to cause a profound loss of biodiversity by indirectly affecting competitive interactions among phytoplankton taxa. Importantly, our results did not show an abrupt loss of biodiversity; instead the reduction in taxa richness levelled off after exceeding a threshold of warming and brownification. These results exemplify the complex nonlinear responses of biodiversity to environmental perturbations and provide further insights for predicting biodiversity patterns to the future warming and brownification of freshwaters.