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Nowadays, infection diseases are one of the most significant threats to humans all around the world. An encouraging strategy for solving this issue and fighting resistant microorganisms is to develop drug carriers for a prolonged release of the antibiotic to the target site. The purpose of this work was to obtain metronidazole-encapsulated chitosan nanoparticles using an ion gelation route and to evaluate their properties. Due to the advantages of the ionic gelation method, the synthesized polymeric nanoparticles can be applied in various fields, especially pharmaceutical and medical. Loading capacity and encapsulation efficiency varFied depending on the amount of antibiotic in each formulation. Physicochemical characterization using scanning electron microscopy revealed a narrow particle size distribution where 90% of chitosan particles were 163.7 nm in size and chitosan-loaded metronidazole nanoparticles were 201.3 nm in size, with a zeta potential value of 36.5 mV. IR spectra revealed characteristic peaks of the drug and polymer nanoparticles. Cell viability assessment revealed that samples have no significant impact on tested cells. Release analysis showed that metronidazole was released from the chitosan matrix for 24 h in a prolonged course, implying that antibiotic-encapsulated polymer nanostructures are a promising drug delivery system to prevent or to treat various diseases. It is desirable to obtain new formulations based on drugs encapsulated in nanoparticles through different preparation methods, with reduced cytotoxic potential, in order to improve the therapeutic effect through sustained and prolonged release mechanisms of the drug correlated with the reduction of adverse effects.