In this work we concentrate on enhancing the antibacterial activity of ZnO nanoparticles by Cr doping, synthesized using a microwave assisted method. The obtained nanoparticles were systematically characterized by X-ray diffraction, Fourier transformation infrared spectroscopy, UV-Vis spectroscopy and field emission scanning microscopy, and the results were correlated. Powder diffraction patterns reveal that Cr is incorporated into the ZnO crystal lattice without disturbing the original wurtzite crystal structure. The vibrational spectra show significant shifts in the peaks with Cr addition, indicating the successful incorporation of Cr ions into ZnO. Optical absorption measurements reveal that the band gap of ZnO decreased from 3.19 to 3.09 eV, which confirmed the existence of a ZnO–Cr catalyst influence interaction. The surface morphology of ZnO changed from hexagonal rod to spherical-hexagonal nanorod structures after Cr doping. Energy dispersive spectral analysis confirms the phase-purity of the synthesized powders. Here, we show that in addition to altering the structure, composition, morphology and optical absorption characteristics of ZnO, the introduction of Cr dopant can also lead to dramatic changes in the antibacterial activity of Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria. The investigated Cr doped ZnO nanocomposite material showed excellent antimicrobial activity compared to ZnO. The bacterial activity was found to be higher for Gram positive bacteria possessing thicker cell walls than that for Gram negative bacteria.