Biological activity of Euglena mutabilis, an acidophilic, photosynthetic protozoan, contributes to the formation of Fe-rich stromatolites in acid mine drainage systems. E. mutabilis is the dominant microbe in bright green benthic mats (biofilm), coating drainage channels at abandoned coal mine sites in Indiana. It builds biolaminates through phototactic and aerotactic behavior, similar to prokaryotes, by moving through precipitates that periodically cover the mats. E. mutabilis also contributes to formation of Fe-rich stromatolites by (1) intracellularly storing Fe compounds released after death, contributing to the solid material of stromatolites and acting as nucleation sites for precipitation of authigenic Fe minerals, and (2) generating O2 via photosynthesis that further facilitates precipitation of reduced Fe, any excess O2 not consumed by Fe precipitation being released to the atmosphere. Recognition of E. mutabilis–dominated biofilm in acidic systems raises a provocative hypothesis relating processes involved in formation of Fe-rich stromatolites by E. mutabilis to those responsible for development of Precambrian stromatolitic Fe formations and oxygenation of the early atmosphere.