The extract of Siegesbeckia pubescens herb and its chemical constituents were tested for the ability to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production in BV2 microglia. The methanol extract and the 90% MeOH fraction of S. pubescens effectively attenuated lipopolysaccharide-induced nitric oxide production. Several steps of chromatography yielded eight ent-kaurane diterpenes (1–8) and one ent-pimarane diterpene (9) from the 90% MeOH fraction. Among these compounds, compounds 2–9 showed significant inhibitory effect on lipopolysaccharide-induced nitric oxide production in BV2 microglia. Compounds 3 and 9 concentration-dependently decreased the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), supported by quantitative real time polymerase chain reaction (PCR) and Western blot analysis. These results suggest that ent-kaurane and ent-pimarane diterpenes isolated from S. pubescens are expected to be potential candidates against neuroinflammation-related disease.