Red blood cells feature remarkable mechanical properties while navigating through microcirculation vessels and during spleen filtration. An unusual combination of plasma membrane and cytoskeleton physical properties allows red blood cells to undergo extensive deformation. Here we used atomic force microscopy multiparametric imaging to probe how cellular organization influences nanoscale and global mechanical properties of cells in both physiological and pathological conditions. Our data obtained in native conditions confirmed that, compared to healthy cells, cells from patients with hereditary spherocytosis are stiffer. Through vertical segmentation of the cell elasticity, we found that healthy and pathological cells display nanoscale architecture with an increasing stiffness along the direction of the applied force. By decoupling the mechanical response of the plasma membrane from its underlying cytoskeleton, we find that both components show altered properties in pathological conditions. Nanoscale multiparametric imaging also revealed lipid domains that exhibit differential mechanical properties than the bulk membrane in both healthy and pathological conditions. Thanks to correlated AFM-fluorescence imaging, we identified submicrometric sphingomyelin-enriched lipid domains of variable stiffness at the red blood cell surface. Our experiments provide novel insights into the interplay between nanoscale organization of red blood cell plasma membrane and their nanomechanical properties. Overall, this work contributes to a better understanding of the complex relationship between cellular nanoscale organization, cellular nanomechanics and how this 3D organization is altered in pathological conditions.