Aligner treatment is associated with bacterial colonization, leading to enamel demineralization. Chitosan nanoparticles have been demonstrated to have antibacterial properties. This in vitro study aims to determine the effect of adding chitosan nanoparticles to directly 3D-printed clear aligner resin with regard to antibiofilm activity, cytotoxicity, degree of conversion, accuracy, deflection force, and tensile strength. Different concentrations (2%, 3%, and 5% w/w) of chitosan nanoparticles were mixed with the clear resin, and the samples were then 3D printed. Additionally, the thermoforming technique for aligner manufacturing was utilized. The obtained specimens were evaluated for antibiofilm activity against Streptococcus mutans bacteria and cytotoxicity against L929 and 3T3 cell lines. Additionally, Fourier transform infrared spectroscopy via attenuated total reflection analysis was used to assess the degree of conversion. Geomagic Control X software was utilized to analyze the accuracy. In addition, the deflection force and tensile strength were evaluated. The results indicated a notable reduction in bacterial colonies when the resin was incorporated with 3 and 5% chitosan nanoparticles. No significant changes in the cytotoxicity or accuracy were detected. In conclusion, integrating biocompatible chitosan nanoparticles into the resin can add an antibiofilm element to an aligner without compromising the material’s certain biological, mechanical, and physical qualities at specific concentrations.