The paper discusses a method to functionalize cotton fabrics using biologically active natural compounds to achieve the antibacterial characteristics required for medical application. The biologically active natural compounds include propolis, beeswax, and chitosan. Three 100 % cotton knitted fabrics with different degrees of compactness were impregnated in the emulsions containing the active ingredients and fabric variant G3 with the highest degree of impregnation was considered for the evaluation of the antibacterial properties and comfort characteristics. The results show that the treated cotton fabric had high antibacterial activity against both gram positive bacteria Staphylococcus aureus and Streptococcus ${eta}$ haemolytic, and gram negative bacteria Escherichia coli and Pseudomonas aeruginosa. The presence of the biologically active natural compounds on the cotton substrates modified the surface of the textile fibers as seen in the SEM images. The treatment also improved fabric comfort properties, the cotton substrates became less air permissive and more hygroscopic after the treatment. The experimental results indicated that propolis, beeswax and chitosan can be applied as an emulsion to functionalize cotton textile materials. The antibacterial performance of the functionalized fabrics suggested that the cotton fabrics treated with those biologically active natural compounds have the potentials to be used in medical fields.