The aim of the current study was to prepare and evaluate the efficacy of novel Azithromycin loaded chitosan nanoparticles. Azithromycin loaded chitosan nanoparticles was prepared by ionic gelation of chitosan with tripolyphosphate anions. The prepared Azithromycin loaded chitosan nanoparticles was characterized by dynamic light scattering, zeta potential, encapsulation efficiency, in-vitro drug release for three hours, and in-vitro antibacterial activity against Escherichia coli. The results of particle size and zeta potential for the prepared Azithromycin loaded chitosan nanoparticles were determined and found to be 276.5 nm and + 25 mV respectively. The encapsulation efficiency result was 80%. The results after 3 hours of dissolution were 53.05 % for Azithromycin loaded chitosan nanoparticles and 7.25 % for azithromycin solution. Furthermore, the Azithromycin loaded chitosan nanoparticles showed broader inhibition zone and higher antibacterial activity against Escherichia coli compared with azithromycin solution. In conclusion, Azithromycin loaded chitosan nanoparticles are a promising platform for enhancing dissolution and antibacterial activity of Azithromycin against Escherichia coli.