The biocidal effect of ZnO nanostructured films was studied using Escherichia coli ATCC 43897. The ZnO nanoparticles were synthesized in diethylene glycol by using zinc acetate forced hydrolysis. X-ray diffraction analysis confirmed the formation of single wurtzite-type ZnO phase with a crystallite size of 20.59 nm. Transmission electron microscopy observations revealed spherical-shaped particles in the nanoscale regime with a mean particle size of 21.96 nm. It was found that the addition of trioctylphosphine during synthesis favored much improved dispersion of ZnO nanoparticles, with smaller particle size; that is, 16.28 nm. Meanwhile, ZnO film grown onto glass substrate by spin-coating revealed single phase with the formation of aggregates (≈700) having mushroom-like morphology formed of very fine particles in the nanoscale regime. The as-deposited nanostructured films exhibited a hydrophilic character. The classical bacteriological and electrochemical impedance spectroscopy measurements enabled the biocidal effect of ZnO nanostructured films with 94% inactivation efficiency after 90 min of contact time. © 2018 Desalination Publications. All rights reserved.