The bacterial inactivation of Escherichia coli on Ag–cotton textiles were investigated under different experimental conditions with novel Ag-nanoparticles fixed on cotton textiles. The evaluation of the bactericide performance of the Ag–cotton was compared with the one observed for Ag–TiO2–cotton loaded textiles. The procedure used allowed us to prepare highly dispersed Ag-cluster species, 2–4 nm in size, as observed by transmission electron microscopy (TEM). By depth profile analysis, the X-ray photoelectron spectroscopy (XPS) revealed that the Ag profile in the 15 topmost layers remained almost constant before and after E. coli inactivation. The cotton loading was fairly low and of the order of 0.10 wt.% Ag/wt. of cotton. By infrared spectroscopy no modification of the Ag–cotton could be detected before or after E. coli inactivation due to the small absorption coefficient of Ag and the very low metal loading of Ag on the cotton. The E. coli was completely abated on Ag–cotton textiles immediately after the contact took place, due to the strong bacteriostatic effect of the dispersed silver clusters on the cotton surface.