Quantifications of phytoplankton biomass and species composition are crucial for monitoring biodiversity and population dynamics in aquatic environments, and both direct microscopic counts and fluorescence-based methods have been widely used for monitoring. Recent advancements in DNA metabarcoding offer an alternative way of easily assessing diversity and species composition. However, a comprehensive comparison of the relative merits and limitations of DNA- and fluorescence-based methods is currently lacking. Here we compare phytoplankton community composition measured via fluorescence and DNA metabarcoding in an outdoor, replicated mesocosm experiment. We show that there is a positive correlation between fluorescence-measured biomass and DNA read and amplicon sequence variants (ASV) numbers for cyanobacteria, but either weak or no correlation for the other phytoplankton groups assessed (cryptophytes, chromophytes, and green algae). In addition, DNA metabarcoding was systematically better at detecting cryptophytes, which were rarely detected via fluorescence. Hence, while DNA metabarcoding may not provide reliable biomass estimates for the majority of phytoplankton groups, metabarcoding analysis offers higher taxonomic resolution and the capability to detect rare phytoplankton groups. Overall, our findings provide new insights into the strengths and limitations of each method and highlight the considerable potential and importance of including DNA barcoding in freshwater ecosystem assessment and biomonitoring programmes with a focus on biodiversity assessments.