Abstract: In this study, the optimal process for the extraction, the antioxidant activity and antibacterial effect of total flavonoids from leaves of Eucalyptus Grandis x E.Urophylla were investigated.The results showed that the optimal conditions for extracting flavonoids were determined as 120min extraction with 60% ethanol at a liquid- solid ratio20∶1 and 65℃. Under the optimal conditions, the extraction rate of total flavonoids was 14.77%. The scavenging capacity of the extracts from leaves of Eucalyptus Grandis x E.Urophylla on DPPH free radicals was up to 94.12%.Besides, strong antibacterial effects of the extracts against common septic and toxin-producing microorganisms in foods were found.