Purpose: To develop ampicillin trihydrate-loaded chitosan nanoparticles by modified ionic gelation method and evaluate their antimicrobial activity. Methods: Ampicillin trihydrate-loaded chitosan nanoparticles were prepared by ionic gelation method with the aid of sonication. Parameters such as the zeta potential, polydispersity, particle size, entrapment efficiency and in vitro drug release of the nanoparticles were assessed for optimization. The antibacterial properties of the nanoparticle formulation were evaluated and compared with that of a commercial formulation (reference). Results: Scanning electron microscopy revealed that the nanoparticles were in the nanosize range but irregular in shape. Concentrations of 0.35 %w/v of chitosan and 0.40 %w/v sodium tripolyphosphate (TPP) and a sonication time of 20 min constituted the optimum conditions for the preparation of the nanoparticles. In vitro release data showed an initial burst followed by slow sustained drug release. The nanoparticles demonstrated superior antimicrobial activity to plain nanoparticles and the reference, due probably to the synergistic effect of chitosan and ampicillin trihydrate. Conclusion: Modified ionic gelation method can be utilized for the development of chitosan nanoparticles of ampicillin trihydrate. Polymer and crosslinking agent concentrations and sonication time are rate-limiting factors for the development of the optimized formulation. The chitosan nanoparticles developed would be capable of sustained delivery of ampicillin trihydrate. © Pharmacotherapy Group.