Aims To investigate inactivation effect and mechanism of zinc oxide nanoparticles (ZnO NPs) activity against Campylobacter jejuni biofilms. Methods and Results ZnO NPs with concentrations of 0, 0.6, 1.2 and 6 mmol l-1 were employed in antimicrobial tests against Camp. jejuni planktonic cells and biofilms. Campylobacter jejuni sessile cells in biofilms were more resistant to a low concentration of ZnO NPs when compared to planktonic cells. The ZnO NPs penetrated the extracellular polymeric substance (EPS) without damage to the EPS and directly interacted with the sessile bacterial cells, as determined using infrared spectroscopy and scanning electron microscopy. Raman spectroscopy shows alterations in quinone structures and damage to nucleic acids following Camp. jejuni treatment with ZnO NPs. The mechanism of DNA damage is most likely due to the generation of reactive oxygen species (ROS). Spectroscopic-based partial least squares regression (PLSR) models could predict the number of surviving sessile cell numbers within a bacterial biofilm (=log 4 CFU, root mean square error of estimation <0.36) from Fourier transform infrared (FT-IR) spectral measurements. Conclusions ZnO NPs were found to have antimicrobial activity against Camp. jejuni biofilms. ZnO NPs penetrated the biofilm EPS within 1 h without damaging it and interacted directly with sessile cells in biofilms. Alterations in the DNA/RNA bases, which are owing to the generation of ROS, appear to result in Camp. jejuni cell death. Significance and Impact of the Study ZnO NPs may offer a realistic strategy to eliminate Camp. jejuni biofilms in the environment.