Escherichia coli, like other gram-negative bacteria, is protected from the surrounding harsh environment by a cell wall consisting of the peptidoglycan and outer membrane. Whereas the cytoplasmic membrane is the selective barrier, the cell wall provides mechanical strength for the cell. As bacteria navigate various environments, osmotic pressure can change dramatically due to changes in local solute concentration. The peptidoglycan together with the cellular proteins mitigates the osmotic stress that would otherwise cause lysis. The mechanical properties of E. coli cells and its individual layers have been largely indeterminable until the recent development of probe-based measurement tools. Since their invention, scientists have reported significant data measuring elasticity, modulus, and stiffness using atomic force microscopy (AFM). Fundamentally, in order to determine these mechanical properties through probe-based techniques, the contact area and load should be well defined. The load can be precisely calculated through the AFM cantilever spring constant. However, the silicon tip contact area can only be estimated, potentially leading to compounding uncertainties. Therefore, we developed a methodology to determine nanomechanical properties of E. coli using a nanoindenter.