Pathogen-derived nucleic acids are crucial signals for innate immunity. Despite the structural similarity between those and host nucleic acids, mammalian cells have been able to evolve powerful innate immune signaling pathways that originate from the detection of cytosolic nucleic acid species, one of the most prominent being the cGAS-STING pathway for DNA and the RLR-MAVS pathway for RNA, respectively. Recent advances have revealed a plethora of regulatory mechanisms that are crucial for balancing the activity of nucleic acid sensors for the maintenance of overall cellular homeostasis. Elucidation of the various mechanisms that enable cells to maintain control over the activity of cytosolic nucleic acid sensors has provided new insight into the pathology of human diseases and, at the same time, offers a rich and largely unexplored source for new therapeutic targets. This Review addresses the emerging literature on regulation of the sensing of cytosolic DNA and RNA via cGAS and RLRs.