Prickly pear Opuntia ficus-indica (L.) Mill.,1768) is a succulent plant belonging to the Cactaceae family, native to Central America but now diffused both in the the Mediterranean area (mainly Sicily, Calabria, Puglia, Sardinia and Malta) and in the temperate areas of America, Africa, Asia and Oceania. The oil obtained from its seeds, rich in polyunsatured fatty acids, has also known antimicrobial and antioxidant properties. Herein we evaluated not only the capacity of this oil to inhibit the formation of biofilm by different pathogens but also to block the metabolic changes taking place in the microbial cells included in the biofilm. The oil was capable to inhibit at 38.75% the biofilm of Escherichia coli just at a concentration of 1 microliter/ml, and, when tested against Pseudomonas aeruginosa, the inhibition reached the 74%. The oil was also effective against Pseudomonas aeruginosa (71.84% of inhibition) and the phytopathogen Pectobacterium carotovorum (63.06%). The metabolic activity of the microbial cells present within the biofilm was also strongly inhibited and, when the oil was tested against P. carotovorum, microbial cell metabolism was completely inhibited. The action of the prickly pear seeds oil was effective also in blocking at 64.97 % the metabolism of Listeria monocytogenes cells, which therefore had conversely demonstrated to be more resistant compared to the other bacteria (31% of biofilm inhibition). Data from the present study indicates a wide field of application of this oil, with interesting potentialities for food and health purposes.