Nanoindentation is commonly used to determine the mechanical properties of the engineering materials. Young’s modulus of a bulk material can be extracted from the load–depth data obtained from an indentation test with a prescribed Poisson’s ratio that is unknown for a new material. The effect of Poisson’s ratio on material’s mechanical property characterization remains unknown. In this paper, finite element analysis was used to simulate nanoindentation testing on specimens of low-carbon steel AISI1018, steel alloy AISI4340, and aluminum alloy 6061T6 with a cylindrical flat-tip indenter. The effects of Poisson’s ratio on measurements of indentation load versus depth curves, Young’s modulus, hardness, and pile-up of the specimens were investigated and formulated. The Poisson ratio ranging from 0 to 0.49 was considered. It was found that the linear part at the beginning of the indentation loading process from the load versus depth curve was proportional to the Young’s modulus and significantly affected by the Poisson’s ratio. The indentation pile-up was also sensitive to the Poisson’s ratio. Combining the formulas from this work with the Hertzian contact equation, the Young’s modulus and the Poisson’s ratio can be determined simultaneously.