A long-chain amino acid containing dipeptide has been found to form a hydrogel in phosphate buffer whose pH ranges from 6.0 to 8.8. The hydrogel formed at pH 7.46 has been characterized by small-angle X-ray scattering (SAXS), wide-angle powder X-ray diffraction (PXRD), Fourier transform infrared (FT-IR) spectroscopy, field-emission scanning electron microscopy (FE-SEM), high-resolution transmission electron microscopy (HR-TEM) imaging and rheological analyses. The microscopic imaging studies suggest the formation of a nanofibrillar three-dimensional (3D) network for the hydrogel. As observed visually and confirmed Theologically, the hydrogel at pH 7.46 exhibits thixotropy. This thixotropic property can be exploited to inject the peptide. Furthermore, the hydrogel exhibits remarkable antibacterial activity against Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, which are responsible for many common diseases. The hydrogel has practical applicability due to its biocompatibility with human red blood cells and human fibroblast cells. Interestingly, this hydrogel shows high resistance toward proteolytic enzymes, making it a new potential antimicrobial agent for future applications. It has also been observed that a small change in molecular structure of the gelator peptide not only turns the gelator into a nongelator molecule under similar conditions, but it also has a significant negative impact on its bactericidal character.