Nano-drug delivery is a promising tactic to enhance the activity and minimize the cytotoxicity of antimicrobial drugs. In the current study, chitosan nanoparticles (CSNPs) were used as a carrier for the delivery of gentamicin sulfate (GM) and ascorbic acid (AA). The particles were synthesized by ionotropic gelation method and characterized by FT-IR, Zeta potential, and transmission electron microscope imaging. The obtained particles were evaluated for their in vitro antimicrobial activity and cytotoxicity. The prepared particles (GM–AA–CSNPs) under the optimal condition of 4:1:1 of chitosan to drug ratio showed encapsulation efficiency and loading capacities of 89% and 22%, respectively. Regarding biological activities, GM–AA–CSNPs showed a lower minimum inhibitory concentration (MIC) than free gentamicin sulfate and GMCSNPs mixture without presenting cytotoxicity against normal cells (HSF). Moreover, the GM–AA–CSNPs did not exhibit hemolytic activity. These results highlight that the GM–AA–CSNPs are confirmed as a hopeful formula for future investigations on the development of antimicrobial preparations.