Using cellulose as raw material, dialdehyde cellulose (DAC) was prepared via selective oxidation with sodium periodate. Single-factor and orthogonal experiments were conducted to optimize the oxidation conditions (sodium periodate concentration, suspension pH, and reaction time) and to evaluate their effects on the aldehyde content and sample retention rate of DAC. The structural characteristics and antibacterial performance of DAC films were also investigated. The results showed that the optimal preparation conditions were sodium periodate concentration of 7%, suspension pH of 3.0, and reaction time of 4.0 h. Under these conditions, the aldehyde content of DAC reached 83%, and the sample retention rate was 80.33%. Meanwhile, The order of influence of each factor on the aldehyde group content is as follows: sodium periodate concentration > reaction time > suspension pH. Fourier transform infrared spectroscopy (FTIR) revealed a new characteristic peak at 1712 cm⁻¹ corresponding to aldehyde groups, confirming successful oxidation. Scanning electron microscopy (SEM) showed increased surface roughness, etching marks, and local damage after oxidation. The DAC film exhibited antibacterial activity against Staphylococcus aureus, Escherichia coli, and Bacillus anthracis, with inhibition zone diameters of 1.50 mm, 8.33 mm, and 6.90 mm, respectively (P < 0.05), whereas the unmodified cellulose film showed no inhibitory effect. These results indicate that oxidation treatment endows DAC with significant antibacterial properties, providing a theoretical basis for the development of food preservative and active packaging materials.