Toll-like receptors (TLRs) belong to pattern recognition receptors, which respond to danger signals such as pathogen-associated molecular patterns or damage-associated molecular patterns. Upon TLR activation in microglia, the major immune cells in the brain, distinct signaling cascades trigger the production of inflammatory molecules, being a critical feature in neuroinflammation and neurodegenerative processes. Recently, individual microRNAs (miRNAs) were shown to act as endogenous TLR ligands. Here, we conducted systematic screening for miRNAs as potential TLR7/8 ligands by small RNA sequencing of apoptotic neurons and their corresponding supernatants. Several miRNA species were identified in both supernatants and injured neurons, and 83.3% of the media-enriched miRNAs activated murine and/or human TLR7/8 expressed in HEK293-derived TLR reporter cells. Among the detected extracellular miRNAs, distinct miRNAs such as miR-340-3p and miR-132-5p induced cytokine and chemokine release from microglia and triggered neurotoxicity in vitro. Taken together, our systematic study establishes miRNAs released from injured neurons as new TLR7/8 activators, which contribute to inflammatory and neurodegenerative responses in the central nervous system (CNS).