The objectives of this research were to develop and characterize edible chitosan film containing lactoferrin as a natural antimicrobial agent, and to investigate the combination effects of lactoferrin with lysozyme in chitosan film against the growth of Escherichia coli O157:H7 and Listeria monocytogenes. Chitosan films containing lactoferrin, lysozyme, or nisin were fabricated, and the antimicrobial concentrations were 0.5, 1, or 2 mg in a circular disc of chitosan film. Three concentrations of lactoferrin or EDTA (0.28, 0.56, or 1.12 mg per disc) were also incorporated into the chitosan film containing lysozyme to investigate the combination effects of lactoferrin. The water barrier properties of the chitosan films containing lactoferrin were characterized. The antimicrobial activities against E. coli O157:H7 and L. monocytogenes were determined using the agar diffusion assay and cell count assay. The chitosan films containing lactoferrin less than 1 mg per disc did not alter the water vapor permeabilit...