Steady developments made in nanotechnology-based products have facilitated new perspectives for combating drug-resistant fungi. Silver nanoparticles represent one of the most attractive nanomaterials in biomedicine due to their exclusive optical, electromagnetic, and catalytic properties and antifungal potency compared with other metal nanoparticles. Most studies show that the physicochemical parameters affecting the antifungal potential of AgNPs include the shape, size, surface charge, and concentration and colloidal state. For the present study, pullulan (P) and its oxidized counterpart (PO) have been selected as matrices for the silver nanoparticles’ generation and stabilization (AgNPs). The TEMPO (2,2,6,6-tetramethylpiperidin-1-yl radical)–sodium hypochlorite–sodium bromide system was used for the C6 selective oxidation of pullulan in order to introduce negatively charged carboxylic groups in its structure. The structure and morphology of the synthesized AgNPs were analyzed using FTIR and EDX. The main objective of this study was to elucidate the antifungal activity of AgNPs on the clinical yeasts isolates and compare the performance of AgNPs with the conventional antifungals. In this study, different concentrations of AgNPs were tested to examine antifungal activity on various clinical isolates.