The objective of the present study was to characterize the essential oil of Myrciaria tenella (MTEO) leaves, to determine the effects of distillation time (1 to 5 h), and to evaluate its action on acute pain and inflammation both in vivo and in vitro. MTEO was obtained by hydrodistillation for 4 h and 36 compounds were detected, mainly consisting of hydrocarbon terpenes such as β-pinene (21.46%), α-pinene (19.43%), E-caryophyllene (10.89%) and aromadendrene (9.17%). A high yield of α-pinene/β-pinene-rich MTEO was obtained after only 2 h of extraction. For in vivo biological assays, MTEO was administered to male Swiss mice, showing an action against hypernociception (acetic acid-induced abdominal writhing, formalin and Von Frey models) and inhibiting carrageenan-induced paw edema. Production of IL-6, TNF-α, IL-1β, PGE2 was reduced and production of IL-10 was increased, with no change in motor performance. MTEO also reduced cell production of NO and cytokines, in addition to inhibiting COX activity.