In this study, oil bodies (OBs) from Gevuina avellana (OBs-G) and Madia sativa (OBs-M) were isolated and characterized. Microscopic inspection revealed that the monolayer on OB-G was thinner compared to that on OB-M. Cytometric profiles regarding size, complexity, and staining for the two OB sources were similar. Fatty acid to protein mass ratio in both OBs was near 29, indicating high lipid enrichment. OBs-G and OBs-M showed a strong electrostatic repulsion over wide ranges of pH (5.5-9.5) and NaCl concentration (0-150 mM). Proteins displaying highly conserved sequences (steroleosins and aquaporins) in the plant kingdom were identified. The presence of oleosins was immunologically revealed using antibodies raised against Arabidopsis thaliana oleosins. OBs-G and OBs-M exhibited no significant cytotoxicity against the cells. This is the first report about the isolation and characterization of OBs-G and OBs-M, and this knowledge could be used for novel applications of these raw materials.