The microbiome, all microorganisms in their natural habitat, can be collected on filters and its DNA extracted. Sequencing of this metagenome allows us to observe microorganisms not under a microscope or in the lab but via their DNA and in their natural habitat, revealing who they are and what they can do. Uncharted microbial sequences from the environment hold potential to change our view of the tree of life, by uncovering a vast microbial diversity that is not easily cultivated in the lab. Novel microorganisms with surprising biological traits await discovery in newly sequenced metagenomes. In addition, environmental sequences published by others are available in public databases, waiting to be further explored. The chapters of this thesis describe the search for unexpected biology in environmental sequences, both in newly sequenced datasets and in those already published. Reanalysis of already published datasets allows for comparisons between microbiomes from many different habitats, and for classical ecological questions to be addressed on the global scale. The chapters also describe the design of algorithms and tools to taxonomically annotate sequences from previously unknown microorganisms that are common in underexplored habitats and still surface in those more intensely studied like human-associated or marine habitats, and to use these annotations for an accurate and comprehensive view of the microbiome. Surprising biology is found deep down in the Black Sea, where elusive bacteria include archaeal-type lipids in their cell membrane, challenging a once-thought fundamental divide across the tree of life in the molecular composition of the membrane. Surprising biology is also found in the discovery of structural colour—the striking display of changing colours depending on the angle of observation—in the bacterial domain and in many different microbial habitats, including curiously the deep ocean where no light penetrates. By reanalysis of thousands of environmental sequencing studies across a wide range of habitats and geographical locations, the social niche breadth of microorganisms—the range of communities in which each lives—is quantified and ecological and genomic correlates of microbial specialism versus generalism are revealed. Together, the chapters paint a picture of a microbial world that is still largely left uncharted. With the development of new algorithms and tools, and the (re)analysis of large-scale data, this thesis uncovers a fraction of that vast microbial unknown.