Short-read, high-throughput sequencing (HTS) methods have yielded numerous important insights into microbial ecology and function. Yet, in many instances short-read HTS techniques are suboptimal, for example, by providing insufficient phylogenetic resolution or low integrity of assembled genomes. Single-molecule and synthetic long-read (SLR) HTS methods have successfully ameliorated these limitations. In addition, nanopore sequencing has generated a number of unique analysis opportunities, such as rapid molecular diagnostics and direct RNA sequencing, and both Pacific Biosciences (PacBio) and nanopore sequencing support detection of epigenetic modifications. Although initially suffering from relatively low sequence quality, recent advances have greatly improved the accuracy of long-read sequencing technologies. In spite of great technological progress in recent years, the long-read HTS methods (PacBio and nanopore sequencing) are still relatively costly, require large amounts of high-quality starting material, and commonly need specific solutions in various analysis steps. Despite these challenges, long-read sequencing technologies offer high-quality, cutting-edge alternatives for testing hypotheses about microbiome structure and functioning as well as assembly of eukaryote genomes from complex environmental DNA samples.