Neuroinflammation is an inflammatory response in the nervous system that is associated with various neurological diseases including Alzheimer's diseases and others. Many studies evaluated the anti-inflammatory potential of Santalum album (S. album) extract, but none of them analyzed its effects against neuroinflammatory response in vitro. In addition, the precise mechanism underlying the anti-inflammatory effect of the extract has not yet been elucidated. Therefore, in this study, we investigated the effect of S. album extract on modulation of toll-like receptor 3 (TLR3) agonist polyinosnic-polycytidylic acid (PolyI:C)-induced neuroinflammatory response in human neuroblastoma cells. The TLR3-mediated immune response was differentially modulated by S. album extract in SH-SY5Y cells. In addition, treatment of cells with the conditioned medium (CM) of S. album extract significantly increased the mRNA levels of IFN-β, IFN-α, MxA and OAS-1 and decreased IL-6, CXCL8, CCL2 and IP-10. S. album extract has indirectly affected the expression of IFNs and inflammatory cytokines in SH-SY5Y cells. Furthermore, the extract was able to modulate PolyI:C-induced inflammatory response in Caco2 cells. Overall, S. album was capable to attenuate PolyI:C-induced neuroinflammatory effect through the induction of TLR2, TLR4 and the modulation of TLR negative regulators of the TRAF3, IRF3 and NF-κB pathways.