Litter-associated microorganisms play key roles in forested streams by decomposing and transferring energy from plant litter to higher trophic levels. To circumvent the shortcomings of traditional microbiological methods and conventional microscopy to assess the composition of microbial communities associated with decomposing litter, a range of DNA-based techniques have been developed in the last 15 years. One of the most promising tools is DNA metabarcoding, which combines DNA-based identification (DNA barcoding) with high-throughput sequencing of environmental samples with unknown species compositions. This chapter describes best practices for sampling and assessing stream fungi and bacteria in mixed communities on decomposing leaf litter by using DNA metabarcoding. DNA is extracted from the litter and the DNA barcodes are amplified by using broad-range primers for fungi and bacteria. The resulting amplicons are subjected to high-throughput sequencing. Finally, the generated sequences are processed in a bioinformatics pipeline, and taxonomic identification is achieved by comparing sample sequence clusters with reference sequences from public databases. Application of the method has been successful in a variety of studies and promises to revolutionize the monitoring of fungi and bacteria on decomposing litter.