Objectives <p>An in vitro study to verify the potential effectiveness of an orthodontic adhesive incorporating a polybetaine zwitterionic mixture in preventing biofilm formation.</p> Materials and methods <p>2-methacryloyloxyethyl phosphorylcholine (MPC) and sulfobetaine methacrylate (SBMA), were added to An adhesive at 1 wt% (MS1) And 3 wt% (MS3). The MS0 group had no zwitterions. Flowability measurement, shear bond strength (SBS), the adhesive remnant index (ARI), and the contact angle were assessed. Bracket bonding was performed, and cross-sections were examined using scanning electron microscopy (SEM). Biofilm formation was analyzed using confocal laser scanning microscopy. Statistical analyses were conducted using R software. Wilcoxon tests with the Holm correction were used for non-parametric data, and pairwise t-tests with the Bonferroni correction were used for parametric data. Significance was set at <i>P</i> &lt; 0.0001.</p> Results <p>Flow analysis showed no significant differences in the experimental groups compared to the MS0 group (<i>P</i> &gt; 0.05). The SEM images confirmed uniform bonding in all groups. SBS decreased significantly with higher MS content (<i>P</i> &lt; 0.0001), And ARI scores shifted with the addition of zwitterionic mixtures, increasing scores to 1 And 2. MS3 showed a significantly lower contact angle compared to MS0 (<i>P</i> &lt; 0.05). MS3 exhibited reduced biofilm formation and lower fluorescence intensity (<i>P</i> &lt; 0.05).</p> Conclusions <p>Despite reductions in SBS, all adhesives remained at minimum acceptable levels. The 3 wt% zwitterionic adhesive effectively suppressed biofilm formation and may help prevent demineralization.</p> Clinical relevance <p>An orthodontic adhesive containing a zwitterionic mixture can satisfy clinical properties and prevent side effects due to biofilm formation.</p>