Antifungal activity of the whole plant extract of Achyranthes aspera Linn., procured from three different locations namely, Himachal Pradesh, Punjab and Haryana, was evaluated using agar well diffusion method. The plants were collected in the month of January-2010. The methanolic extract of Achyranthes aspera Linn. (Family: Amaranthaceae) was tested for antifungal activity against various fungal spp. viz. Aspergillus flavus, Aspergillus nidulans, Fusarium and various isolates of Aspergillus niger. Qualitative analysis of phytochemicals showed presence of sugars, proteins, alkaloids, tannins, phenols, saponins, triterpenoids, glycosides and steroids in Achyranthes aspera. The plant showed maximum antifungal activity at 10000µg ml-1 concentration against Aspergillus flavus, Aspergillus nidulans and Fusarium species with the zone of inhibition upto 17.5mm, 16mm and 15.5 mm respectively from Punjab region. In case of Aspergillus niger, maximum zone of inhibition was observed in the isolate grown on pomegranate peels (15 mm). Among the three locations the best antifungal activity was obtained from the plants procured from Punjab region. The result obtained in the present study suggests the significant scope of developing novel broad spectrum antifungal formulations.