Plants of the genus Croton have been used extensively in the northeast of Brazil for treating various clinical conditions. Previous studies have demonstrated that the essential oil of some specimens of Croton sp. have a relaxing effect on tracheal smooth muscle. Our study aimed to characterize the effects of Croton sonderianus essential oil samples, collected at 1:00 pm (EO-13) and 9:00 pm (EO-21), on rat tracheal smooth muscle. The two samples were submitted to gas chromatography (GC) and mass spectrometry (MS) analysis to identify their components. Rat tracheal smooth muscle strips were used to assess the biological activity. The major constituents of EO-21 were: spathulenol (18.32%), beta-caryophyllene (14.58%) and caryophyllene oxide (8.54%) and the major constituents of EO-13 were bicyclogermacrene (16.29%), beta-phellandrene (15.42%) and beta-caryophyllene (13.82%). These samples showed an antispasmodic effect on tracheal smooth muscle strips pre-contracted with high K+ concentration (80 mM) or with acetylcholine. EO-21 increased baseline tonus while EO-13 provoked a decrease. These results demonstrated that EO-13 and EO-21 have different chemical composition and showed myorelaxant activity. In conclusion, EO-13 and EO-21 may have potential therapeutic use in the treatment of bronchospasm.