This study is an attempt to investigate the feasibility of alkali pre-treatment to activate surface hydroxyl groups of cellulose fibers in order to enhance the deposition efficiency of silver nanoparticles (AgNPs) onto cotton fabrics. Cotton samples were pre-treated with various alkali solutions containing different earth metal hydroxides (LiOH, NaOH, and KOH). The as-prepared samples were then treated with aqueous silver nitrate followed by reduction treatment with aqueous ascorbic acid, which caused in situ formation of AgNPs on fiber surfaces. The surface structure of the fabrics was characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD) analysis, and colorimetric data. The amount of silver was measured by using inductively coupled plasma-optical emission spectrometer (ICP-OES). Antimicrobial activity was measured against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria. It was established that alkali pre-treatment had a substantial effect on the formation and adsorption of AgNPs on the fibers. Alkali pre-treated samples were homogeneously coated by AgNPs with high surface coverage. Alkali type had significant effect not only on the amount of AgNPs on the surface but also on its size. High antibacterial activity against both Gram-positive and Gram-negative strains was also demonstrated, even after 10 cycles washing.