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Abstract Titanium dioxide is a photocatalyst, known not only for its ability to oxidize organic contaminants, but also for its antimicrobial properties. In this article, significant enhancement of the antimicrobial activity of TiO₂(up to 32 times) was demonstrated after its activation by ball milling. The antimicrobial activity was analyzed for one fungal and 13 bacterial ATCC strains using the microdilution method and recording the minimum inhibitory concentration (MIC) values. In order to further investigate the correlation between the mechanical activation of TiO₂and its antimicrobial activity, the structure, morphology and phase composition of the material were studied by means of Electron Microscopy, X‐ray diffraction and nitrogen adsorption‐desorption measurements. UV‐Vis diffuse reflectance spectra were recorded and the Kubelka‐Munk function was applied to convert reflectance into the equivalent band gap energy (E_g) and, consequently, to investigate changes in theE_gvalue. X‐ray photoelectron spectroscopy was used to analyze the influence of mechanical activation on the Ti 2p and O 1s spectra. The presented results are expected to enable the development of more sustainable and effective advanced TiO₂‐based materials with antimicrobial properties that could be used in numerous green technology applications.