Well-preserved microfossils occur in abundance through more than 1000 m of lower Mesoproterozoic siliciclastic rocks composing the Roper Group, Northern Territory, Australia. The Roper assemblage includes 34 taxa, five interpreted unambiguously as eukaryotes, nine as possible eukaryotes (including Blastanosphaira kokkoda new genus and new species, a budding spheromorph with thin chagrinate walls), eight as possible or probable cyanobacteria, and 12 incertae sedis. Taxonomic richness is highest in inshore facies, and populations interpreted as unambiguous or probable eukaryotes occur most abundantly in coastal and proximal shelf shales. Phylogenetic placement within the Eukarya is difficult, and molecular clock estimates suggest that preserved microfossils may belong, in part or in toto, to stem group eukaryotes (forms that diverged before the last common ancestor of extant eukaryotes, or LECA) or stem lineages within major clades of the eukaryotic crown group (after LECA). Despite this, Roper fossils provide direct or inferential evidence for many basic features of eukaryotic biology, including a dynamic cytoskeleton and membrane system that enabled cells to change shape, life cycles that include resting cysts coated by decay-resistant biopolymers, reproduction by budding and binary division, osmotrophy, and simple multicellularity. The diversity, environmental range, and ecological importance of eukaryotes, however, were lower than in later Neoproterozoic and Phanerozoic ecosystems.