Cholesterol is an important component of total lipid in mammalian cellular membranes; hence, the knowledge of its association with lipid bilayer membranes will be essential to understanding membrane structure and function. A droplet interface bilayer (DIB) provides a convenient and reliable platform through which values for permeability coefficient and activation energy of water transport across the membrane can be extracted. In this study, we investigated the effect of acyl chain structure in amphiphilic monoglycerides on the permeability of water across DIB membranes composed of cholesterol and these monoglycerides, where the acyl chain length, number of double bonds, and the position of double bond are varied systematically along the acyl chains. To elucidate the role of cholesterol in these membranes, we investigated its influence on water permeability and associated activation energies at two different cholesterol concentrations. Our systematic studies show dramatic sensitivity and selectivity of specific interaction of cholesterol with the monoglyceride bilayer having structural variations in acyl chain compositions. Our findings allow us to delineate the exquisite interplay between membrane properties and structural components and understand the balanced contribution of each.