Real-time elastography (RTE) is a noninvasive imaging modality where tumor-associated changes in tissue architecture are recognized as increased stiffness of the lesion compared to surrounding normal tissue. In contrast to this macroscopic appraisal, quantifying stiffness properties at the subcellular level by atomic force microscopy (AFM) reveals aggressive cancer cells to be soft. We compared RTE and AFM profiling of the same breast lesion to explore the diagnostic potential of tissue elasticity at different length scales. Patients were recruited from women who were scheduled for a biopsy in the outpatient breast clinic of the University Hospital Basel, Switzerland. RTE was performed as part of a standard breast work-up. Individual elastograms were characterized based on the Tsukuba elasticity score. Additionally, lesion elasticity was semiquantitatively assessed by the strain ratio. Core biopsies were obtained for histologic diagnosis and nanomechanical profiling by AFM under near-physiological conditions. Bulk stiffness evaluation by RTE does not always allow for a clear distinction between benign and malignant lesions and may result in the false assessment of breast lesions. AFM on the other hand enables quantitative stiffness measurements at higher spatial, i.e., subcellular, and force resolution. Consequently, lesions that were false positive or false negative by RTE were correctly identified by their nanomechanical AFM profiles as confirmed by histological diagnosis. Nanomechanical measurements can be used as unique markers of benign and cancerous breast lesions by providing relevant information at the molecular level. This is of particular significance considering the heterogeneity of tumors and may improve diagnostic accuracy compared to RTE.