Biofilms can be undesirable, as in those covering medical implants, and beneficial, such as when they are used for waste treatment. Because cohesive strength is a primary factor affecting the balance between growth and detachment, its quantification is essential in understanding, predicting, and modeling biofilm development. In this study, we developed a novel atomic force microscopy (AFM) method for reproducibly measuring, in situ, the cohesive energy levels of moist 1-day biofilms. The biofilm was grown from an undefined mixed culture taken from activated sludge. The volume of biofilm displaced and the corresponding frictional energy dissipated were determined as a function of biofilm depth, resulting in the calculation of the cohesive energy. Our results showed that cohesive energy increased with biofilm depth, from 0.10 ± 0.07 nJ/μm3 to 2.05 ± 0.62 nJ/μm3. This observation was reproducible, with four different biofilms showing the same behavior. Cohesive energy also increased from 0.10 ± 0.07 nJ/μm 3 to 1.98 ± 0.34 nJ/μm3 when calcium (10 mM) was added to the reactor during biofilm cultivation. These results agree with previous reports on calcium increasing the cohesiveness of biofilms. This AFM-based technique can be performed with available off-the-shelf instrumentation. It could therefore be widely used to examine biofilm cohesion under a variety of conditions.