Carboxybetaine methacrylate (CBMA), which has anti-biofouling properties, was incorporated into poly (methyl methacrylate) (PMMA) to prevent multi-species biofilm formation without impairing the critical mechanical properties of the PMMA. The incorporation of 2-methacryloyloxyethyl phosphorylcholine (MPC, 3 wt%) was used as a positive control. PMMA wettability was found to increase with increasing CBMA content. The addition of CBMA resulted in decreased mechanical properties of PMMA. The incorporation of 3% CBMA into PMMA resulted in considerable long-term anti-biofouling effects that reduce adhesion of various fungi, bacteria (Candida albicans, Actinomyces naeslundii, and Veillonella parvula), and a human saliva-derived biofilm. The anti-biofouling effect of CBMA-incorporated PMMA surpassed that of the positive control. By calculating the intensity of the collective band using Raman spectroscopy, CBMA was proven to form the most stable hydration layer. Neither CBMA nor MPC groups showed cytotoxicity. Occlusal splints fabricated using 3% CBMA showed clinically acceptable aesthetic properties with slight turbidity. Despite a limitation in terms of deteriorations of the mechanical properties, the addition of an appropriate amount of CBMA to PMMA inhibited the attachment of fungi and bacteria and suppressed multi-species biofilm growth. The aesthetic properties of CBMAincorporated PMMA are acceptable for clinical practice.