In this work we focus on enhancing the antibacterial activity of ZnO nanoparticles by Mn doping, synthesized using a wet-chemical method. The as-obtained precursor powders were deeply investigated by thermal analyses correlated with the evolved gas analysis (TG-DTA-FT-IR) and by in situ high-temperature XRD to elucidate the thermally induced processes and to understand the manganese doped ZnO nanoparticles formation. The hexagonal wurtzite-type structure and the morphological characteristics of the thermally treated samples have been investigated by X-ray diffraction, and HRTEM. An average particle size ranging between 10 and 29 nm and a polyhedral and spherical morphology with a tendency to form aggregates were evidenced by TEM images. Optical absorption measurements reveal that the band gap of ZnO decreased from 3.19 to 2.99 eV, which confirmed the existence of Zn-O-Mn interaction. The incorporation of the Mn ions into the ZnO lattice has been studied by EPR spectroscopy and also, the generation of reactive oxygen species (ROS) has been evidenced by using the EPR coupled with the spin trapping probe technique. Here, we report that in addition to altering the crystallite size, morphology and optical absorption characteristics of ZnO, the introduction of Mn dopant also improves the antibacterial efficiency against pathogenic microorganisms, namely Escherichia coli and Bacillus cereus.