Although a lot of antibacterial finishes are available for cotton, the user safety and durability of the finish are always the important issues. Some of the natural dyes are known to possess antibacterial properties; however, most of them are nonsubstantive. Hence, they are used in conjunction with eco-friendly natural mordants. Although metallic mordants are effective, they are environmentally pollutants, hence not desirable. In the current work, the novel natural mordant like chitosan with different concentrations (10% and 20% of weight of fiber) was applied on cotton and fabric was printed with natural dyes such as catechu, turmeric, and marigold using screen printing method. All the three dyes were used in powder form in print paste with different concentrations (1%, 5%, and 10%) and guar gum was used as thickener. The printed samples using chitosan as a mordant showed the equivalent color values to that of prints obtained using alum as a mordant. The printed fabric showed excellent antibacterial activity against both gram-positive and gram-negative bacteria. The method may be considered suitable for eco-friendly printing and antibacterial finishing of textile materials.