This study was performed to evaluate the in vitro antimicrobial and antioxidant activities and the phenolic profile of Eucalytus globulus Labill. and Corymbia ficifolia (F. Muell.) K.D. Hill & L.A.S. Johnson leaves. Both leave extracts contain significant amounts of phenolic compounds, mainly flavonoids. Qualitative and quantitative analyses of the phenolic compounds were performed using a HPLC/MS method. The main flavonoid was hyperoside and its highest amount was found in E. globulus (666.42 ± 5.02 μg/g dw plant material). Regarding the flavonol profile, myricetin was the dominant compound and its highest amount was found in C. ficifolia leaves (124.46 ± 0.24 μg/g dw plant material). The antioxidant activity was evaluated by DPPH, TEAC, hemoglobin ascorbate peroxidase activity inhibition (HAPX) and inhibition of lipid peroxidation catalyzed by cytochrome c assays, revealing an important antioxidant potential for both species. In the antimicrobial assays, C. ficifolia extract was found to be more active than E. globulus against both Gram-positive and Gram-negative bacterial strains with the exception of Bacillus subtilis. The results of the present study provide new valuable data regarding the bioactivities of these medicinal species.