Nanomechanical properties of cells could be considered as cellular biomarkers. The main method used to access the mechanical properties is based on nanoindentation measurements, performed with an operator manipulated Atomic Force Microscope (AFM) which is time-consuming and expensive. This is one of the reasons that prevent the transfer of AFM technology into clinical laboratories. In this paper we report a methodology which includes an algorithm (transferred to a script, executed on a commercial AFM) able to automatically move the tip onto a single cell and through several cells to record force curves combined with a smart strategy of cell immobilization. Cells are placed into microwells of a microstructured polydimethylsiloxane (PDMS) stamp. Inside a classical 100 × 100 μm2 AFM field, 100 cells can be immobilized. In an optimal configuration we were able to measure, within 4 h, a population of 900 Candida albicans cells both native and caspofungin treated, which represents an unprecedented performance. We discovered that the population is heterogeneous and can be divided, on the basis of nanomechanical properties, into 2 subgroups.