This study aimed to evaluate the herbicidal activity of ethyl acetate leaf extract of Ageratum conyzoides L. at different subfractions on Amaranthus spinosus L. The leaves of A. conyzoides were sequentially extracted with n-hexane and ethyl acetate respectively and fractionated by chromatography column. The extracts were applied to A. spinosus in pot assays at a concentration of 5%, 10% and 15%. We applied A synthetic herbicide (2,4-D at 0.686 kg a.i. ha−1) for positive control and distilled water for negative control. The A. conyzoides extracts strongly differed in their effect on weed control, shoot and root dry weight and root length of A. spinosus. The most inhibition on A. spinosus growth caused by application of ethyl acetate of A. conyzoides extracts subfraction A by 10% concentration can cause 100% destruction and subfraction B were 95% which both of them cause strongest death on A. spinosus compared with synthetic herbicide (2, 4-D) (23.33%) at 1 Day After Application, while subfraction C and D were not effective. Main constituents identified by GC-MS in subfraction A extract were tetradecanoic acid, ethyl ester (10.26%), precocene II (9.39%), octadecanal (8.23%), 9,12,15-octatadecatrienoic, methyl ester (7.32%), 10-heneicosene (c,t) (5.19%) and neophytadiene (5.09%); in subfraction B were 1-octadecyne (38.57%), phytol (11.24%), di-tert-utylphosphine-d (5.17%) and 1-hexadecine (4.08%); in subfraction C were allobarbital (8.53%), octadecanal (12.69%), and bannamurpanin (26.01%) and octadecanal (30.52%), bannamurpanin (24.06%), 1,8-cineole (15.75%), trans-dodec-5enal (12.28%) and phytol (8.26%) in subfraction D. The ethyl acetate extract subfraction A and B concentration 10% proved the promising control agent against A. spinosus.