Physical attributes of nanoparticles synthesized for therapeutics are often studied as they are known to influence in vitro cellular and in vivo tissue uptake as well as their circulation. Surface chemistry of nanoparticles with biologics is known to control their physicochemical attributes including thermal, magnetic and optical but not nanomechanical properties. For specific bio-application, a wide variety of biologics have been immobilized on the nanoparticle surface. Using atomic force microscopy (AFM), here we present a comprehensive analysis of morphology and nanomechanical properties of gold nanoparticles (GNPs) conjugated with biologics, viz, nonapeptides and poly-ethylene glycol. We have optimized the applied force involved in the AFM measurements to avoid any structural alteration in biologics present on GNPs’ surface. The vertical dimensions of GNPs from AFM studies were in agreement with the hydrodynamic size obtained from Zetasizer. We also monitored that surface chemistry of GNPs significantly influence their nanomechanical attributes including Young’s modulus, adhesion, work of adhesion and surface free energy. The results from these studies demonstrate that surface alteration of GNPs shift the paradigm of their nanomechanical signatures.