The aim of the study was to evaluate in vitro antioxidant and antifungal activities of the ethanolic extract and its fractions from Ocimum gratissimum leaves. The ethanolic extract was obtained by maceration in ethanol and subsequent fractionation with solvents of increasing polarity (hexane, dichloromethane, ethyl acetate and butanol). The Minimum Inhibitory Concentration (MIC) was determined for the ethanol extract and dichloromethane fraction. The antioxidant capacity was evaluated by DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) and ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) free radical scavenging methods, and by FRAP (Ferric Reducing Antioxidant Power). The in vitro antifungal effect was determined by the agar diffusion method on Aspergillus sp. and Rhizopus sp. fungi associated with corn and bean seeds during storage. The best samples with antifungal effect were determined by gas chromatography-mass spectrometry (GC/MS). The ethanolic extract had strong antioxidant capacity for all tested methods (DPPH 371.10±2.98 μg ml-1, ABTS 182.43±1.10 μg ml-1, FRAP 262.39±3.61 TEAC). Regarding the antifungal activity, the ethanolic extract and dichloromethane fraction resulted in total suppression (100%) of fungal growth and MIC ranged from 0.625 to 1.25 mg ml-1. In the GC/MS analysis, 22 substances were detected in all samples evaluated, with predominance of eugenol. These results indicated high biological potential of this plant as a biofungicide