The terrestrial subsurface is estimated to be the largest reservoir of microbial life on Earth. However, the subsurface also harbours economic, industrial and environmental resources, on which humans heavily rely, including diverse energy sources and formations for the storage of industrial waste and carbon dioxide for climate change mitigation. As a result of this anthropogenic activity, the subsurface landscape is transformed, including the subsurface biosphere. Through the creation of new environments and the introduction of substrates that fuel microbial life, the structure and function of subsurface microbiomes shift markedly. These microbial changes often have unintended effects on overall ecosystem function and are frequently challenging to manage from the surface of the Earth. In this Review, we highlight emerging research that investigates the impacts of anthropogenic activity on the terrestrial subsurface biosphere. We explore how humans alter the constraints on microbial life in the subsurface through drilling, mining, contamination and resource extraction, along with the resulting impacts of microorganisms on resource recovery and subsurface infrastructure.