Neuraminidase (NA) is a sialidase present, among various locations, in the envelope/membrane of some bacteria/viruses (e.g., influenza virus), and is involved in infectiveness and/or dispersion. The administration of NA within the brain lateral ventricle represents a model of acute sterile inflammation. The relevance of the Toll-like receptors TLR2 and TLR4 (particularly those in microglial cells) in such process was investigated. Mouse strains deficient in either TLR2 (TLR2 ) or TLR4 (TLR4 ) were used. NA was injected in the lateral ventricle, and the inflammatory reaction was studied by immunohistochemistry (IBA1 and IL-1β) and qPCR (cytokine response). Also, microglia was isolated from those strains and in vitro stimulated with NA, or with TLR2/TLR4 agonists as positive controls (P3C and LPS respectively). The relevance of the sialidase activity of NA was investigated by stimulating microglia with heat-inactivated NA, or with native NA in the presence of sialidase inhibitors (oseltamivir phosphate and N-acetyl-2,3-dehydro-2-deoxyneuraminic acid). In septofimbria and hypothalamus, IBA1-positive and IL-1β-positive cell counts increased after NA injection in wild type (WT) mice. In TLR4 mice, such increases were largely abolished, while were only slightly diminished in TLR2 mice. Similarly, the NA-induced expression of IL-1β, TNFα, and IL-6 was completely blocked in TLR4 mice, and only partially reduced in TLR2 mice. In isolated cultured microglia, NA induced a cytokine response (IL-1β, TNFα, and IL-6) in WT microglia, but was unable to do so in TLR4 microglia; TLR2 deficiency partially affected the NA-induced microglial response. When WT microglia was exposed in vitro to heat-inactivated NA or to native NA along with sialidase inhibitors, the NA-induced microglia activation was almost completely abrogated. NA is able to directly activate microglial cells, and it does so mostly acting through the TLR4 receptor, while TLR2 has a secondary role. Accordingly, the inflammatory reaction induced by NA in vivo is partially dependent on TLR2, while TLR4 plays a crucial role. Also, the sialidase activity of NA is critical for microglial activation. These results highlight the relevance of microbial NA in the neuroinflammation provoked by NA-bearing pathogens and the possibility of targeting its sialidase activity to ameliorate its impact.