The CHARMM-GUI Membrane Builder is a web-based tool designed to facilitate the construction of all-atom protein/membrane or membrane-only systems for molecular dynamics simulations. This tool has been enhanced with new features and improvements to robustly build realistic biological membrane systems. Key enhancements include the addition of over 180 lipid types, such as phosphoinositides, cardiolipin, sphingolipids, bacterial lipids, and ergosterol, an improved lipid packing procedure around proteins, a reliable algorithm for detecting lipid tail penetration into ring structures and protein surfaces, a distance-based algorithm for faster ion placement, CHARMM inputs for P21 image transformation, and NAMD equilibration and production inputs. These improvements are demonstrated by building and simulating a membrane model of the polar and septal regions of the E. coli membrane, which contains five lipid types. The robustness and versatility of the CHARMM-GUI Membrane Builder are highlighted, making it a valuable tool for understanding the structure and dynamics of proteins and lipids in realistic biological membrane environments.The CHARMM-GUI Membrane Builder is a web-based tool designed to facilitate the construction of all-atom protein/membrane or membrane-only systems for molecular dynamics simulations. This tool has been enhanced with new features and improvements to robustly build realistic biological membrane systems. Key enhancements include the addition of over 180 lipid types, such as phosphoinositides, cardiolipin, sphingolipids, bacterial lipids, and ergosterol, an improved lipid packing procedure around proteins, a reliable algorithm for detecting lipid tail penetration into ring structures and protein surfaces, a distance-based algorithm for faster ion placement, CHARMM inputs for P21 image transformation, and NAMD equilibration and production inputs. These improvements are demonstrated by building and simulating a membrane model of the polar and septal regions of the E. coli membrane, which contains five lipid types. The robustness and versatility of the CHARMM-GUI Membrane Builder are highlighted, making it a valuable tool for understanding the structure and dynamics of proteins and lipids in realistic biological membrane environments.