Introduction: Recently, silver nanoparticles were indicated for root canal irrigation. The cytotoxicity evaluation of these agents helps to ascertain confident clinical use. This article aims at evaluating the cytotoxic effect of fungal-derived nanosilver particle irrigant on human periodontal ligament fibroblast (hPDLF) cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Materials and Methods: The test “nanoparticle” irrigant was produced using the fungi “Fusarium semitectum.” The cells were cultured in Dulbecco's Modified Eagle's Medium solution and adjusted to 5 × 103 cells/ml. About 100 μl of cells were seeded into a 96 well microplate and 100 μl test irrigant ranging from 5 to 640 μg/ml concentrations was added to a microplate and incubated at 37°C, 5% CO2 humidified conditions for 24 h. Untreated cells were used as a control group. About 5 mg/ml MTT was added to the plates and incubated at 37°C in 5% CO2 conditions for 4 h. The viability of cells and the percentage inhibition of cell were determined. Results: The 50% inhibition of cells was found to be around 320 μg/ml concentration. Cytotoxicity was found to be dose dependent and increased with higher concentrations of the nanosolution. Conclusion: Fungal-derived nanosilver irrigant is safe to hPDLF cells “in vitro” at a concentration of <320 μg/ml.