Lymphatic filariasis is caused by infection with the parasitic filarial nematodes Wuchereria bancrofti, Brugia malayi and B. timori, transmitted by mosquitoes. The lack of an adulticidal drug poses a challenge to filariasis elimination, hence it is essential to develop an effective antifilarial drug which could either kill or permanently sterilize the adult worms. In the reported work the in vitro activity of a methanolic extract of fruits of Trachyspermum ammi (Apiaceae) against adult bovine filarial Setaria digitata worms has been investigated. A bioassay-guided fractionation was carried out by subjecting the crude extract to flash chromatography. HPLC analysis was done for the crude extract and active fraction. The crude extract and the active fraction showed significant activity against the adult S. digitata by both a worm motility and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction assays. The isolated active principle was chemically characterized by IR, (1)H-NMR and MS analysis and identified as a phenolic monoterpene. It was screened for in vivo antifilarial activity against the human filarial worm B. malayi in Mastomys coucha, showing macrofilaricidal activity and female worm sterility in vivo against B. malayi. The findings thus provide a new lead for development of a macrofilaricidal drug from natural products.