The present work demonstrates a rational designing and synthesis of cholesterol-based amino acid containing hydrogelators with the aim to improve the biocompatibility of these amphiphilic molecules. A thorough structure-property correlation of these hydrogelators was carried out by varying the hydrophilic terminal from a neutral amine to a quaternized ammonium chloride. The amphiphiles having a cationic polar head as the hydrophilic domain and cholesterol as the hydrophobic unit showed better water gelation efficiency (minimum gelation concentration (MGC) similar to 0.9-3.1%, w/v) than the analogous free amines. Presumably, the additional ionic interactions for the quaternized amphiphiles might have played the crucial role in gelation as counterions also got involved in hydrogen bonding with solvent molecules. Hence, the attainment of desired hydrophilic-lipophilic balance (HLB) of hydrophobic cholesterol in combination with the appropriate hydrophilic terminal led to the development of efficient hydrogels. Microscopic investigations revealed the formation of various supramolecular morphologies of hydrogels due to the variation in the molecular structure of the amphiphile. Spectroscopic investigations showed the involvement of hydrogen-bonding, hydrophobic, and pi-pi interactions in the self-assembled gelation. Importantly, biocompatibility of all the cholesterol-based hydrogelators tested against human hepatic cancer derived HepG2 cells showed increased cell viability than the previously reported alkyl-chain-based amphiphilic hydrogelators. In order to incorporate broad spectrum antibacterial properties, silver nanoparticles (AgNPs) were synthesized in situ within the hydrogels using sunlight. The amphiphile-AgNP soft nanocomposite exhibited notable bactericidal property against both Gram-positive and Gram-negative bacteria.