Traditionally, cell surface properties have been difficult to study at the subcellular level, especially on hydrated, live cells. Here, we demonstrate the ability of chemical force microscopy to map the hydrophobicity of single live cells with nanoscale resolution. After validating the technique on reference surfaces with known chemistry, we probe the local hydrophobic character of two medically important microorganisms, Aspergillus fumigatus and Mycobacterium bovis, in relation with function. Applicable to a wide variety of cells, the chemically sensitive imaging method presented here provides new opportunities for studying the nanoscale surface properties of live cells and for understanding their roles in mediating cellular events.