Carboxybetaine methacrylate (CBMA), which has anti-biofouling properties, was incorporated into poly(methyl methacrylate) (PMMA) to prevent multi-species biofilm formation without impairing the criticalmechanical 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 increasingCBMA content. The addition of CBMA resulted in decreased mechanical properties of PMMA. Theincorporation of 3% CBMA into PMMA resulted in considerable long-term anti-biofouling effects thatreduce adhesion of various fungi, bacteria (Candida albicans, Actinomyces naeslundii, and Veillonellaparvula), and a human saliva-derived biofilm. The anti-biofouling effect of CBMA-incorporated PMMAsurpassed that of the positive control. By calculating the intensity of the collective band using Ramanspectroscopy, CBMA was proven to form the most stable hydration layer. Neither CBMA nor MPC groupsshowed cytotoxicity. Occlusal splints fabricated using 3% CBMA showed clinically acceptable aestheticproperties with slight turbidity. Despite a limitation in terms of deteriorations of the mechanicalproperties, the addition of an appropriate amount of CBMA to PMMA inhibited the attachment of fungiand bacteria and suppressed multi-species biofilm growth. The aesthetic properties of CBMAincorporatedPMMA are acceptable for clinical practice.