Our aims of the research were to study the antimicrobial effect of dimethylaminododecyl methacrylate (DMADDM) modified denture base resin on multi-species biofilms and the biocompatibility of this modified dental material. Candida albicans (C. albicans), Streptococcus mutans (S. mutans), Streptococcus sanguinis (S. sanguinis), as well as Actinomyces naeslundii (A. naeslundii) were used for biofilm formation on denture base resin. Colony forming unit (CFU) counts, microbial viability staining, and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) array were used to evaluate the antimicrobial effect of DMADDM. C. albicans staining and Real-time PCR were used to analyze the morphology and expression of virulence genes of C. albicans in biofilm. Lactate dehydrogenase (LDH) array and Real-time PCR were conducted to examine the results after biofilm co-cultured with epithelial cell. Hematoxylin and eosin (HE) staining followed by histological evaluation were used to study the biocompatibility of this modified material. We found that DMADDM containing groups reduced both biomass and metabolic activity of the biofilm significantly. DMADDM can also inhibit the virulence of C. albicans by means of inhibiting the hyphal development and downregulation of two virulence related genes. DMADDM significantly reduced the cell damage caused by multi-species biofilm according to the LDH activity and reduced the expression of IL-18 gene of the cells simultaneously. The in vivo histological evaluation proved that the addition of DMADDM less than 6.6% in denture material did not increase the inflammatory response (p > 0.05). Therefore, we proposed that the novel denture base resin containing DMADDM may be considered as a new promising therapeutic system against problems caused by microbes on denture base such as denture stomatitis.