Objectives: This in vitro study was purposed to examine the antibiofilm activity, weight change, and surface properties including glucose sorption, and roughness of novel nano-ZrO2 incorporated denture liners. Materials and Methods: Modified nano-ZrO2 were added to silicone-based and acrylic resin-based prosthetic lining materials at two different concentrations (0.5% and 1%). The antibiofilm potentials of test groups against Candida albicans (C. albicans), Staphylococcus aureus (S. aureus), and Streptococcus mutans (S. mutans) were determined using 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium (MTT) method. Surface roughness, weight change, and glucose sorption of denture liners were also evaluated after modifying them with nano-ZrO2. Results: According to the antibiofilm activity results, 1% nano-ZrO2 addition to silicon-based and acrylic resin- based denture liners resulted in 16.48% and 13.39% of biofilm inhibition for S. aureus, respectively. 1% nano- ZrO2 addition to silicon-based denture liners also inhibited the S. mutans biofilm formation at an 8.16% rate. Nano-ZrO2 addition to the test groups had no inhibition effect on C. albicans biofilm formation. Surface roughness decreased significantly once nano-ZrO2 was added in acrylic resin-based test groups; however, addition of 0.5% nano-ZrO2 increased silicone-based test group significantly. Conclusions: To mitigate microbial biofilm problems caused by the use of denture liners addition of nano-ZrO2 might be a promising method owing to its antibiofilm capacities especially against biofilms of S. aureus and S. mutans.