In this study, cellulase pre-treated mercerized cotton (EMC) fabrics were treated with citric acid (CA), butane tetra carboxylic acid (BTCA) and nano TiO2 (NTO) to produce fabric with enhanced cross-linking, self-cleaning, and antibacterial properties. However treatment of EMC fabrics with NTO/CA/BTCA has not been reported. The NTO particles were stabilized on the cotton surface using CA/BTCA cross-linking agents. In order to create optimum dry crease recovery angle (DCRA), self-cleaning based on (ΔE*) and bacteria reduction against Gram-positive and Gram-negative bacteria, the appropriate models were obtained based on Design of Expert software. The roles of CA, BTCA and NTO concentrations in cross-linking, self-cleaning, and antibacterial properties of the EMC fabric were investigated using response surface methodology (RSM). Overall, the cellulase pre-treatment of the mercerized cotton fabrics improved the antibacterial activity against Staphylococcus aureus and Escherichia coli bacteria, and also enhanced DCRA and self-cleaning properties significantly as compared with control samples. Also, reflectance spectra analyses (200–400 nm) and Scanning Electron Microscopy (SEM) were employed to confirm the NTO particles on the EMC fabric surface.