Consumers demand more alternatives of riskless antibacterial agents to prevent microbial contamination in food industry. Oxidized carbohydrate may be a potential option as new antibacterial agent. However, the relatively weak antibacterial property of oxidized carbohydrate is not satisfactory. In this paper, dialdehyde β-cyclodextrins with different oxidation degree were prepared by periodate oxidation and their antibacterial properties were systematically studied. The results showed that multi-aldehyde groups were successfully introduced into β-cyclodextrin molecules by periodate oxidation. The aqueous solubility and stability of dialdehyde β-cyclodextrins were improved as expected. It is interesting that dialdehyde β-cyclodextrins possessed outstanding antibacterial activity against both Gram-positive and Gram-negative bacteria. The minimal inhibitory concentrations against E. coli, S. aureus and B. subtilis reached 0.63, 1.25 and 0.63 mg/mL, respectively. Moreover, dialdehyde β-cyclodextrins effectively inhibited bacterial growth on the surface of apples. The results demonstrated that oxidized oligosaccharide with multi-aldehyde groups and good dispersibility in aqueous solution possessed satisfactory antibacterial activity, which can be used as new antibacterial agent in food industry.