Herein we used gelatin, which is an organic, non-toxic, safe and inexpensive substance as antibacterial material for cotton fabrics. There are some studies deals with the topic of finishing of textile materials with gelatin, its antibacterial, antioxidant and anti-fungal activity and its used as bio-material for textiles finishing but these studies neglect the effect of gelatin antibacterial itself which increase our attention to study the antibacterial effect of gelatin itself without any harmful side effects. Pad-dry-cure method was used for cotton fabric which was treated with gelatin in the presence of ammonium dihydrogen phosphate as a catalyst and dimethyloldihydroxyethelyene urea as a cross linker. Factors affecting this treatment were studied. Fourier Transform Infrared spectroscopy (FTIR), physico-mechanical properties, thermal gravimetric analysis and scanning electron microscope analysis were used to characterize the treated cotton fabrics. Disk diffusion method used to assessment antibacterial activity of the treated fabrics. Cytotoxicity of gelatin compared with. ciprofloxacin in viable A549 cells was evaluates by using MTT Results shows that treated cotton fabrics show excellent antibacterial activity towards tested bacteria and there is no observed toxic effects on mammalian cells so that gelatin can be used as safe antibacterial finishing agents of cotton fabrics.