Chitosan properties can be finely tuned for various bio-applications by cross-linking with suitable cross-linkers and the extent of its cross-linking. The effect of concentration of cross-linker on the biopolymer would exhibit different physiochemical properties, surface charges, thermal stability, morphology and their swelling behavior. In this study, chitosan has been cross-linked with varying concentration of hydrophilic terephthaldehyde (TAL) as cross-linker and characterized using FT-IR, XRD and thermogravimetric analysis (TGA). The effect of concentration of the cross-linker on the morphology, surface charge, pH dependent swelling was studied. The formation of imine bond between chitosan and TAL was confirmed by using FT-IR. The XRD analysis shows increase in crystallinity upon increasing the cross-linker concentration of TAL in comparison with the pure chitosan. The thermal stability of the hydrogels was also studied. With increase in the concentration of TAL, the hydrogels show a polyphasic structure and the surface charge become more positive. The TAL cross-linked chitosan exhibited both highest and lowest in swelling at the physiological pH with varying concentration of the cross-linker. The chitosan-terephthaldehyde hydrogels exhibits good antimicrobial response towards gram negative bacteria. The results indicate chitosan-terephthaldehyde hydrogels (CST) to have potential applications in various biomedical applications at physiological pH.