© 2022 SAABOpuntia ficus-indica (L.) Mill., which is generally consumed as a fruit, has many pharmacological properties. In this study, we investigated antibacterial and antiproliferative effect of methanol extract of Opuntia ficus-indica (L.) Mill peel. and swarming motility and biofilm formation on Pseudomonas aeruginosa PAO1. Also, the phenolic profile was analyzed by HPLC and rutin was found to be as major compound. The mode of action for the in vitro antiquorum sensing and antiproliferative activity of the extract was elucidated by qRT-PCR and molecular docking, respectively. Our results indicated that the methanol extract of Opuntia ficus-indica (L.) Mill. peel extract significantly decreased the expression level of quorum sensing related lasI, lasR, rhlI and rhIR genes. According to the antiproliferative activity studies, the extract was found to be more cytotoxic activity against breast (MDA-MB-231) and liver (HepG2) cancer cell lines compared to colon (DLD-1) cancer cell line with IC50 values of 2.00 ± 0.19, 3.85 ± 0.24 and 26.17 ± 0.73, respectively. Additionally, the antiproliferative activity against breast cancer could be the result of the inhibition of CCL18 by rutin.