Few studies are concerned about the essential oil extracted from leaves of Eucalyptus benthamii Maiden et Cambage that shows high content of α-pinene. The goal of this paper was to investigate the in vitro effect of the essential oil of E. benthamii and α-pinene on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse peritoneal macrophages. Macrophages were harvested by washing with phosphate buffered saline and cultured with 10 μg/mL LPS. Three concentrations (5, 10, and 20 μg/mL) of the essential oil of E. benthamii and α-pinene were investigated. Nitrite levels were measured based on the Griess reaction, an indirect assay for NO production. The essential oil of E. benthamii significantly suppressed NO production in murine peritoneal macrophages at 10 and 20 μg/mL. In contrast, α-pinene did not inhibit NO production.