Candida albicans is an emerging multidrug-resistant opportunistic pathogen representing an important source of invasive disease in humans and generating high healthcare costs worldwide. The development of a rapid and simple antifungal susceptibility test (AFST) could limit the spread of this disease, increase the efficiency of treatment, and lower the risk of developing resistant strains. We developed a microfluidic chip containing an array of microwells that were designed to trap the cells and perform rapid antifungal susceptibility tests using optical nanomotion detection (ONMD). Yeast cell entrapment in a microwell allows for a very rapid exchange of growth medium with the antifungal, which enables performing single-cell ONMD measurements on the same cell before and after antifungal treatment. The exposure to a low concentration of the antifungal caspofungin or flucanozole induced a significant decrease in the nanomotion signal, demonstrating the high sensitivity of this technique. We used this chip to quantify the real-time response of individual C. albicans cells to the antifungal treatment in as fast as 10 min. This simple and label-free technique could be further developed into a simple-to-use device that allows the performance of fast AFST as part of a routine hospital procedure in developed and also eventually developing world countries.