Objective The objective of this in vitro study was to comparatively evaluate the antifungal and physical properties of tissue conditioner incorporated with nanoparticles (NPs) of different types and concentrations. Materials and methods A total of 198 tissue conditioner samples were used in this study. The samples were categorized into a control group, namely, tissue conditioner without NPs (Group 1), and test groups, namely, tissue conditioner incorporated with zinc oxide (ZnO) NPs (Group 2) and magnesium oxide (MgO) NPs (Group 3). The antifungal properties and surface roughness of the samples were evaluated. The groups were further subdivided into seven subgroups: control (without NPs), 5% ZnO NPs, 10% ZnO NPs, 15% ZnO NPs, 3% MgO NPs, 5% MgO NPs, and 7% MgO NPs by weight. Surface roughness was measured using an optical profilometer, and antifungal activity was measured in terms of the diameter of the inhibition zone (DIZ) using the well diffusion method over seven days. Results The result showed that the 5% ZnO NPs subgroup had the lowest mean surface roughness, whereas the 15% ZnO NPs subgroup had the highest antifungal activity. Increasing the concentration of NPs increased the antifungal property, and there was a steady decrease in DIZ from day one to day seven in all test groups. Conclusion Our results showed that the incorporation of various concentrations of ZnO and MgO NPs into tissue conditioner samples positively affected their physical and antifungal properties. The highest antifungal activity was found in the 15% ZnO NPs subgroup, and the lowest surface roughness was found in the 5% ZnO NPs subgroup.