Background: Eschweilera nana Miers is a tree widely distributed in Cerrado, Brazil. Objective: In this study, we aimed to describe its phytochemical properties and antioxidant and topical anti inflammatory effects for the first time, as well validate an high performance liquid chromatography with ultraviolet/visible (HPLC UV Vis) method for the separation and quantification of the main components (hyperoside and rutin) in the hydroalcoholic extract of E. nana leaves. Materials and Methods: Structural identification of compounds in E. nana extract was performed by analysis of spectral data by 1H nuclear magnetic resonance, 13C nuclear magnetic resonance and/or ESI/EM. The HPLC UV Vis method was validated according International Conference on Harmonization (ICH) parameters. The 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) method were used for determination of in vitro antioxidant activities and the croton oil induced inflammation for evaluation of in vivo anti inflammatory effects. Results: Hyperoside, rutin, α-amirin, β-amirin, β-sitosterol, and stigmasterol were identified in the hydroalcoholic extract of E. nana leaves. HPLC UV Vis was validated according to ICH parameters. Furthermore, in vitro and in vivo assays demonstrated that the hydroalcoholic extract and methanol fraction showed significant antioxidant and topical anti inflammatory effects, as they were able to reduce ear edema induced by croton oil application. Conclusions: This research showed the first phytochemical study of E. nana extract and their biological activities may be associated with the presence of flavonoids in the extracts.