Abstract Humiria balsamifera is used in traditional medicine as anthelmintic, expectorant, to treat hepatitis, diarrhea, hemorrhoids; to cure chronic wounds; and to alleviate toothaches. This species occurs in Jurubatiba shoal, Rio de Janeiro state-Brazil, a rich region which offers a variety of promising bioactive product sources. The present study focuses on the chemical and pharmacological evaluation of H. balsamifera. The n-hexane, dichloromethane and ethyl acetate leaf fractions exhibited higher inhibitory potential on NO production. Friedelin (1), quercetin (2) and quercetin-3-α-O-arabinopyranoside (3) were isolated and characterized; the latter is described for the first time for H. balsamifera. Quercetin (2) showed the best inhibitory activity on NO production and moderate inhibition of TNF-α production. These results contribute to the knowledge of Humiria balsamifera as a source of anti-inflammatory compounds. Furthermore, the identification of the terpenes ß-amyrone, betulin, citronellol, eremophillene, dihydroactinolide and borneol, and the isolation of quercetin-3-α-O-arabinopyranoside are being reported for the first time for this species.