We present the fabrication of specialized nanotools, termed nanoscalpels, and their application for nanolithography and nanomechanical manipulation of biological objects. Fabricated nanoscalpels have the shape of a thin blade with the controlled thickness of 20–30 nm and width of 100–200 nm. They were fabricated using electron beam induced deposition at the apex of atomic force microscope probes and are hard enough for a single cut to penetrate a ∼45 nm thick gold layer; and thus can be used for making narrow electrode gaps required for fabrication of nanoelectronic devices. As an atomic force microscope-based technique the nanoscalpel provides simultaneous control of the applied cutting force and the depth of the cut. Using mammalian cells as an example, we demonstrated their ability to make narrow incisions and measurements of local elastic and inelastic characteristics of a cell, making nanoscalpels also useful as a nanosurgical tool in cell biology. Therefore, we believe that the nanoscalpel could serve as an important tool for nanofabrication and nanosurgery on biological objects.