Abstract Structural, microstructural and bactericidal surface properties of TiO 2 ‐coated glass substrates elaborated by reactive Radiofrequency sputtering are investigated. As pathogenic bacteria in biofilms are a major concern in food industries due to their growing resistance to cleaning and sanitizing procedures, the development of photoactive surfaces exhibiting bactericidal properties is acknowledged as an effective approach to tackle bacterial contaminations. Our principal aim concerns the study of the photoactive top‐layer thickness impact (from 80 nm to ~500 nm) on Listeria monocytogenes . Structural characterization of the TiO 2 layers demonstrates that anatase and rutile phases are both present, depending on the film thickness. Photocatalytic activity of the samples has been evaluated through the degradation of aqueous methylene blue ( MB ) solutions under UVA light illumination for various time periods. The results show an efficiency rating increase according to TiO 2 film thickness up to a threshold value close to 400 nm. Moreover, a significant decrease of the adherent bacteria number is observed after 20 min of UVA illumination. The quantitative study of the bactericidal activity associated with scanning electron microscopy observations of the postprocess bacteria damaged cells demonstrates the efficiency of the 240‐nm‐thick TiO 2 coating sample. The results are correlated with the production of hydroxyl radicals during the process of photocatalysis.