Asymmetry between outer and inner leaflets of cell membrane, such as variations in phospholipid composition, cholesterol (CHOL) distribution, stress levels, and ion environments, could significantly influence the biophysical properties of membranes, including the lateral organization of lipids and the formation of membrane nanodomains. To elucidate the effects of lipid component, lipid number mismatch, CHOL concentration asymmetry, and ionic conditions on membrane properties, we constructed several sets of all-atom, multi-component lipid bilayer models. Using molecular dynamics (MD) simulations, we investigated how membrane asymmetry modulates its biological characteristics. Our results indicate that CHOL concentration, whether symmetric or asymmetric between the leaflets, is the primary factor affecting membrane thickness, order parameters of the lipid tail, tilting angles of lipid molecules, water permeability, lateral pressure profiles, and transmembrane potential. Both low and high CHOL concentrations significantly alter lipid bilayer properties. Inducing cross-leaflet stress by mismatching lipid numbers can modify lipid order parameters and the tilting angles but has only mild effect on lateral pressure profiles and membrane thickness. Additionally, we found that transmembrane potential, generated by ions concentration differences across the membrane, can influence water permeability. Our findings expand the current understanding of lipid membrane properties and underscore the importance of considering CHOL and phospholipid asymmetry in membrane biophysics. The membrane models developed in our study also provide more physiological conditions for studying membrane proteins using MD simulations.