Public concerns about food safety have triggered a worldwide implementation of new legislations aimed at banning many of the most popular food conventional antifungal treatments. There is therefore an urgent need to identify novel and safer solutions to prevent fungal contamination of food. The antifungal effect of zinc oxide nanoparticles (ZnO NPs) against the postharvest pathogenic fungus Penicillium expansum has been investigated in this study. An automated turbidimetric assay, with a standard 96-well microplate, has been developed and optimised regarding the selection of the inoculum size in order to collect sequential optical density measurements. Data were processed by the updated version of the Lambert Pearson model to estimate the minimum inhibitory concentration and the non-inhibitory concentration values which were found to be 9.8 and 1.8 mM (i.e. 798 and 147 ppm), respectively. The current results show that turbidimetry is a reliable technique for assessing the antifungal activity of metal nanoparticles and that zinc oxide (ZnO) is an effective fungicide which can be potentially used to control food safety.