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Atomic Force Microscopy (AFM) is a powerful tool enabling the mechanical characterization of biological materials at the nanoscale. Since biological materials are highly heterogeneous, their mechanical characterization is still considered to be a challenging procedure. In this paper, a new approach that leads to a 3-dimensional (3D) nanomechanical characterization is presented based on the average Young’s modulus and the AFM indentation method. The proposed method can contribute to the clarification of the variability of the mechanical properties of biological samples in the 3-dimensional space (variability at the x–y plane and depth-dependent behavior). The method was applied to agarose gels, fibroblasts, and breast cancer cells. Moreover, new mathematical methods towards a quantitative mechanical characterization are also proposed. The presented approach is a step forward to a more accurate and complete characterization of biological materials and could contribute to an accurate user-independent diagnosis of various diseases such as cancer in the future.