[Objective] The study aims to explore the antibacterial activity and allelopathy of flavonoids from leaves of Eleutherococcus brachypus, and to provide the basis for the development of plant-derived antimicrobial agents and their allelopathy in plant protection and agricultural production. [Methods] Its leaves as raw materials, the flavonoids were extracted from E. brachypus leaves by ultrasonic-assisted extraction, the antibacterial ability of leaf flavonoids extract against Staphylococcus aureus, Bacillus subtilis, and Salmonella were evaluated by the antibacterial sphere, minimum antibacterial concentration (MIC), in the meantime, the allelopathic effects of leaf flavonoids extract were studied by measuring the apoptosis rate, malondialdehyde (MDA) content in apoptosis cell, and root and bulbil growth inhibition rate of onion epidermal cells under stress of different concentrations (100%, 50%, 25%) the flavonoids extracts from E. brachypus. [Results] (1) The yield of leaf flavonoids from E. brachypus was 42.3 mg/g. (2) Under the same concentration, the antibacterial activity of leaf flavonoids against the gram-positive bacteria (S. aureus and B. subtilis) was significantly higher than that of the gram-negative bacterium (P<0.05), and the antibacterial activities were proportional to the concentration of leaf flavonoids extracts of E. brachypus. The maximum antibacterial coil diameters of the leaf flavonoids extracts of E. brachypus against S. aureus, B. subtilis, and Salmonella were 24.4, 22.1, 18.3 mm, respectively, and the minimum inhibitory concentration of S. aureus, B.subtilis, and Salmonella were 1.31, 2.63, 5.25 mg/mL, respectively. Considering the antibacterial coil diameter and MIC of the three tested species, the antibacterial ability was successively decreased as: S. aureus, B. subtilis, Salmonella. (3) Different concentrations of leaf flavonoids from E. brachypus could induce apoptosis of the upper epidermal cells of Allium cepa L., and many morphological characteristics from apoptotic cells in A. cepa L. were examined under a light microscope, such as, cytoplasmic breakdown and collapse, many cytoplasmic vesicles formation, at the same time, the nuclear membrane fractured and even disappeared, chromatin condensation and edging, finally, the apoptosis rate and MDA content in epidermal cells were also increased with treatment concentration. (4) Moreover, the growth of root and bulbil of onion bulb were inhibited under different concentrations of leaf flavone extracts treatment, and the relative inhibition rate of root and bulbil growth of onion bulb was also concentration-dependent, compared to the control. In conclusion, leaf flavonoids of the E. brachypus had antibacterial activity, apoptosis-inducing of plant cells, and inhibiting the growth of roots and bulbil of A. cepa L. These findings suggest that the leaf flavonoids extract from E. brachypus had certain antibacterial activity, and it also had some allelochemicals phytotoxic effects on apoptosis-induction epidermal cell of A. cepa L., and growth inhibition of root and bulbil of onion bulb by inducing membrane lipid peroxidation.