In the present study, we have synthesized silver nanoparticles using Azadirachta indica (A. indica) skin extract as a reducing agent and characterized it. The antifungal activity of the silver nanoparticles was assessed against three different clinical isolates of T. asahii. The silver nanoparticles showed an efficient antifungal activity against T. asahii and, it was again established by time kill assay and Post-AntiFungal Effect (PAFE). Synergistic effect of silver nanoparticles with fluconazole or nystatin revealed a Minimum Inhibitory Concentration (MIC) decrease by 4-fold for both drugs studied and inhibited biofilm formation. An in vivo antifungal efficacy of silver nanoparticles using female Balb/C mice model revealed a mean 4log reduction of CFU in kidney when treated with 90mg/kg. Morphology changes of T. asahii cells, treated with silver nanoparticles, were observed using SEM. Cell membrane damage was quantified from the treated cells using flow cytometry by a Propidium Iodide (PI) uptake assay. The mechanism of action of the new silver nanoparticles against T. asahii was interpreted, and we learnt that they directly bind to ergosterol. Further, silver nanoparticles were not cytotoxic to normal cells (NIH 3T3) and hemocompatible. We concluded that the new silver nanoparticles exhibited promising antifungal activity against T. asahii.