In continuous research for bioactive compounds obtained from plants to use for weed control in sustainable agriculture, the aerial parts of Cachrys pungens Jan (Umbelliferae) were extracted with methanol and then fractionated using hexane, chloroform (CHCl₃) and ethyl acetate (AcOEt). The potential phytotoxicity of total methanolic extract and each fraction was assayed in vitro on seed germination and root elongation of lettuce (Lactuca sativa L.) and the most active fractions were assayed on three of the most common weeds (Lolium perenne, Amaranthus retroflexus, Echinochloa crus-galli). Non linear regression that allowed to obtain the ED₅₀ index for both physiological processes was applied. The fraction bioassays indicated the following hierarchy of phytotoxicity for both processes: CHCl₃ ≥ AcOEt > hexane. Moreover, in the present work was chemically characterized for the first time (through HPTLC) the polar fraction of this species pointing out the high presence of flavonoids and phenolic acids. In particular six of them have been chemically characterized and quantified (naringin, quercetin, catechin, caffeic acid, ferulic acid and gallic acid). These results make C. pungens Jan a potential source of natural compounds employable for an eco-friendly agriculture.