There is a growing realization that endodontic infections are often polymicrobial, and may contain Candida spp. Despite this understanding, the development of new endodontic irrigants and models of pathogenesis remains limited to mono-species biofilm models and is bacterially focused. The purpose of this study was to develop and optimize an interkingdom biofilm model of endodontic infection and use this to test suitable anti-biofilm actives. Biofilms containing Streptococcus gordonii, Fusobacterium nucleatum, Porphyromonas gingivalis, and Candida albicans were established from ontological analysis. Biofilms were optimized in different media and atmospheric conditions, prior to quantification and imaging, and subsequently treated with chlorhexidine, EDTA, and chitosan. These studies demonstrated that either media supplemented with serum were equally optimal for biofilm growth, which were dominated by S. gordonii, followed by C. albicans. Assessment of antimicrobial activity showed significant effectiveness of each antimicrobial, irrespective of serum. Chitosan was most effective (3 log reduction), and preferentially targeted C. albicans in both biofilm treatment and inhibition models. Chitosan was similarly effective at preventing biofilm growth on a dentine substrate. This study has shown that a reproducible and robust complex interkingdom model, which when tested with the antifungal chitosan, supports the notion of C. albicans as a key structural component.