Zootherapy is a traditional secular practice among the Guarani-Kaiowá indigenous ethnic group living in Mato Grosso do Sul, Brazil. My people use the oil extracted from larvae of the snout beetle Rhynchophorus palmarum (Linnaeus, 1758) to treat and heal skin wounds and respiratory diseases. Based on this ethnopharmacological knowledge, the chemical composition and antioxidant, antimicrobial, and healing properties of R. palmarum larvae oil (RPLO) were investigated, as well as possible toxic effects, through in vitro and in vivo assays. The chemical composition of the RPLO was determined using gas chromatography coupled with mass spectrometry. The antioxidant activity of RPLO was investigated through the direct 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, and the antimicrobial activity was evaluated against Gram-positive and Gram-negative bacteria that are pathogenic to humans. The healing properties of RPLO were investigated by performing a cell migration assay using human lung fibroblasts (MRC-5), and the toxicity was analyzed, in vivo, using a Caenorhabditis elegans model and MRC-5 cells, in vitro. RPLO contains 52.2% saturated fatty acids and 47.4% unsaturated fatty acids, with palmitic acid (42.7%) and oleic acid (40%) representing its major components, respectively. RPLO possesses direct antioxidant activity, with a half-maximal inhibitory concentration (IC50) of 46.15 mg.ml-1. The antimicrobial activity of RPLO was not observed at a concentration of 1% (v/v). RPLO did not alter the viability of MRC-5 cells and did not exert toxic effects on C. elegans. Furthermore, MRC-5 cells incubated with 0.5% RPLO showed a higher rate of cell migration than that of the control group, supporting its healing properties. Taken together, RPLO possesses direct antioxidant activity and the potential to aid in the healing process and is not toxic toward in vitro and in vivo models, corroborating the safe use of the oil in traditional Guarani-Kaiowá medicine.