Microbial growth and colonization are one of the disadvantages after the long-term use of denture soft liners. This study was aimed to evaluate the effect of the addition of magnesium oxide nanoparticles (MgONPs) on the antimicrobial activity of a denture soft liner. A total of 84 samples were prepared from a soft denture liner and divided into two groups, control group and test group to which 3% by weight of MgONPs were added, antimicrobial tests for Candida albicans, Staphylococcus aureus, and a mixture of the biofilm of both microorganisms were done, cell density count and adherence assay test was recorded before and after 6 months storage in distilled water. The results showed that the modified group performed a better antimicrobial activity compared to the control group, the addition of 3% of the nanofiller showed a significant reduction in the mean value of growth of both C. albicans, S. aureus, and the biofilm mixture (P < 0.05) before and after the storage period. In conclusion, the addition of 3% by weight of MgONPs to the denture soft liner significantly suppressed the growth of C. albicans, S. aureus up to 6 months.