A series of dialdehyde microcrystalline cellulose (DAMC) particles crosslinked with EPL (EPL-DAMCs) was successfully prepared by reacting DAMC with varying amounts of e-Poly-L-lysine (EPL). Based on the excellent antibacterial property of e-poly-L-lysine (EPL), the obtained EPL-DAMCs have a broad-spectrum antibacterial activity, and their physicochemical and antibacterial activities were also investigated. Fourier -transform infrared spectroscopy results demonstrated the formation of Schiff base between the aldehyde groups in DAMC and amino groups of EPL. The DAMC particles showed an aggregated structure of fibres. Among all DAMC crosslinked with EPL (EPL-DAMCs), the EPL-DAMC-4, with a lysine content of 0.46 ± 0.08 mg/g, showed the highest antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Salmonella typhimurium, with the minimum inhibitory concentration (MIC) of 3.75, 15.0, 7.5 and 3.75 mg/mL, respectively. Compared with DAMC, the EPL-DAMC-4 exhibited the better inhibition effect and antimicrobial stability on the tested strains. These findings suggested that EPL-DAMCs might be used as antimicrobial biomaterial and have great potential in food packaging field.