Soft denture liners have limitations like short lifespan and increased microbial buildup. Despite promise as a non-leaching antimicrobial polymer in dentistry, the impact of dimethylaminododecyl methacrylate (DMADDM) on soft liner performance remains unexplored. This study aimed to evaluate the effect of integrating different concentrations of DMADDM to cold cure acrylic resin soft liner, on its antimicrobial activity, cytotoxicity, and physical properties. The same properties were compared to a conventional commercially available denture soft liner. The study employed a control group (conventional soft liner) and three test groups containing 3.3%, 6.6%, and 10% (total mass fraction) DMADDM, respectively. Antimicrobial activity against Candida albicans and Streptococcus mutans was assessed through colony counts and biofilm biomass. Cytotoxicity was evaluated using an oral epithelial cell line. Additionally, wettability and hardness were measured to assess physical properties. Incorporation of DMADDM significantly reduced Candida albicans and Streptococcus mutans counts, and biofilm biomass, compared to the control. Additionally, DMADDM improved the soft liner's wettability and mitigated long-term hardness increase. In conclusion, DMADDM holds promise in enhancing soft liner performance. However, careful selection of its optimum concentration is crucial to ensure both safety and efficacy for future clinical use.