Background: Pseudomonas aeruginosa causes infections in human particularly immunocompromised patients with cystic fibrosis, severe burns and HIV, resulting in high morbidity and mortality. The pathogenic bacteria P aeruginosa produces virulence factors regulated by the mechanism called quorum sensing system. Objective: The aim of this study was to assess the anti-quorum sensing activity of Ageratum conyzoides extracts Method: Chromobacterium violaceum reporter strain CV026 was used to highlight any interference with bacterium QS and strains derived from P. aeruginosa PAO1 were used to reveal any interference with the expression of quorum sensing genes, and to assess any impact of extract on the kinetics of the production of pyocyanin, elastases and biofilm formation. Results: Hydro-methanolic extract at the sub-inhibitory concentration of 100 μg/mL reduced quorum sensing virulence factors production such as, pyocyanin, elastases, rhamnolipids and biofilm formation in P. aeruginosa PAO1 after 18 hours monitoring. Extract showed significant inhibition in HSL-mediated violacein production on C. violaceum CV026 after 48 hours monitoring. Biofilm formation was inhibited up to 32%. It affected QS gene expression in PAO1. The regulatory genes lasR / rhlR and the lasI synthases were most affected. At 8hours, hydro-methanolic extract reduced both QS gene to more than 30% (lasI/lasR and rhlI/R respectively 33.8% /30.2% and 36% /33.2%). RhlA and lasB genes have been relatively affected (13.4% and 28.9%). After 18 h, this extract reduced significantly the expression of regulatory 30 genes lasR (31%) and rhlR (39.6%) although synthases genes seemed to be less affected (lasI/21.2% and rhlI/11.6%). A limited impact was observed on the downstream genes (lasB /20.0% and rhlA /15.3%). No negative impact was observed on CV026 and PAO1 growth and cell viability. Our study also showed that A. conyzoides having ample amount of phenolics, flavonoids and triterpenoids. This phytochemical content could be one of the factors for showing anti quorum potential. Conclusion: Results indicate that hydro methanol 80 % extract from A. conyzoides could be a source of potential QS inhibition compounds.