Hydrophobicity of microorganisms is an important surface property that allows them to adhere to a variety of abiotic surfaces but also to other cells. In that sense it can be a determining factor in the pathogenicity of microorganisms, or on their capacity to form biofilms on surfaces. However, measuring the hydrophobic properties of complex surfaces such as living cells using classic methods (water contact angle measurements, adhesion to hydrocarbons) is usually a challenge. In this practical, we will demonstrate how atomic force microscopy and its microfluidic version, FluidFM, can be used to measure and quantify these hydrophobic properties at the micro- and nano-meter scales in liquid. For that, the first steps will be to immobilize living microalgae (Parachlorella kessleri) and yeasts (Saccharomyces cerevisiae) on functionalized surfaces. Then to evaluate the global hydrophobicity of cells, a hydrophobic probe such as an air bubble formed at the aperture of a microfluidic AFM cantilever using FluidFM, can be used to probe the cell surface in force spectroscopy experiments. In this case, the bubble formed is micrometer-sized, and will allow to quantify the global hydrophobic properties of a given cell. But if the goal is for example to reveal nanoscale hydrophobic structures on cells, a smaller hydrophobic probe is needed. In this case, a gold AFM tip can be functionalized using a CH3-terminated thiol to make it hydrophobic, and used also in force spectroscopy experiments. Because the edge of the tip that will be in contact with the cell surface is of a few nanometers, information at the nanometer-scale can be collected. This practical is open to any scientists interested in cell surface hydrophobicity, and competences in AFM or biophysics are not required.