Studies on the isolation of soil-born fungi and their phytotoxicity against weeds and millet were conducted in the Centre of Biotechnology and Microbiology at the University of Peshawar in 2015. For this purpose, soil from the three botanical gardens in Peshawar valley – the Botanical Garden of the Pakistan Forest Institute (PFI), the Islamia College Botanical Garden and the University of Peshawar Botanical Garden in Aza Khel – were collected and analyzed for the identification and isolation of different species of soil-born fungi. A total of seven species of fungi were isolated from the three botanical gardens, 5 species (Alternaria spp., Aspergillus niger, Aspergillus flavus, Penicillium spp., and Rhizopus spp.) from the botanical garden of the Pakistan Forest Institute, 4 species (Fusarium spp., Cladosporium spp., Penicillium spp., and A. flavus) from Islamia College Botanical Garden and 3 species (A. flavus, Alternaria spp and A. niger) from the University of Peshawar botanical garden in Aza Khel. Among these, A. flavous was found in all three botanical gardens with higher frequency such as in PFI (90%), in Islamia College Botanical Garden (65%) and the University of Peshawar Botanical Garden in Aza Khel (50%). This species produces a large number of colonies such as in PFI (56), Islamia College (24) and in the University of Peshawar (7) colonies per petri dish. In the second part of the studies, crude extract of A. flavus was collected and tested on the seed mortality of five different weeds and millet. Those weeds were A. retroflexus, C. album, Sonchus arvensis, Galium aparine and Viola arvensis. Crude extract of A. flavus with different concentrations (i.e., 10, 100 and 1000 μg/mL) were prepared and applied on weed seeds kept in the petri dishes. Due to the toxic effect of the extract of A. flavus, all weed seeds were killed, i.e., A. retroflexus (100%), C. album (100%), S. arvensis (100%), G. aparine (100%) and V. arvensis (100%). Similarly crude extract of A. flavus also killed millet seeds completely (100%). Therefore, our results suggest that extract of A. flavus could be used as a biological control agent for controlling weeds through targeted spray as well as in turf and commercials areas.