We report a novel approach to probe the interior of single mammalian and bacterial cells at nanometer resolution by combining Focused Ion Beam (FIB) and Atomic Force Microscopy (AFM). After removing a finely controlled thin layer on the target cells with FIB milling, AFM cantilever tips functionalized were employed to measure the hydrophobic forces at interior regions of the cells under aqueous environments. The hydrophobic forces acquired from the nucleus and cytoplasm regions of COS-7 cells were compare, and it was revealed that the nucleus regions were more hydrophobic possibly due to the exposed lipids from the nucleus membrane. The interior of E.coli were heterogeneous, whereas the peptidoglycan and outer membrane regions were clearly shown. The FIB serial milling performed on single E.coli enabled a faster AFM spatial mapping and reconstruction of intracellular architectures. The exposed cell slices were also accessible to the Secondary Ion Mass Spectrometry (SIMS), where the SIMS image confirmed richer carbon elements in the cell cytoplasm.