Prickly pear fruit peel constitutes a high percentage of the fruit and could be a natural, economic agro-industrial waste of potential use in the nutraceutical industry. This study aimed to isolate and characterize the main constituents of the fruit peel and evaluate its antibacterial activity. A methanol extract was successively fractionated using hexane, chloroform and ethyl acetate. The n-hexane fraction was evaluated for its fatty acid content using gas chromatography mass spectrometry (GC-MS), revealing linolenic acid (omega-3) as the major fatty acid (60.56%), while an ethyl acetate fraction was analyzed using ultra-performance liquid chromatography electrospray tandem mass spectrometry (UPLC-ESI-MS/MS), resulting in the identification of 6 phenolic acids and 9 flavonoids, where caffeic acid (43.69%) and quercetin (14%) were found the most abundant. The ethyl acetate fraction was subjected to column chromatography, resulting in the isolation of four flavanols, viz. astragalin (1), quercetin 5,4'-dimethyl ether (2), isorhamnetin-3-O-glucoside (3) and isorhamnetin (4). Antibacterial evaluation revealed that the EtOAc fraction is the most potent active fraction against the selected pneumonia pathogens, and quercetin 5,4'-dimethyl ether (2) is the most active among the isolated compounds. Virtual docking of the isolated compounds showed promising in silico anti-quorum sensing efficacy, indicating that they could represent natural antibacterial agents. These findings indicate that the unused waste from prickly pear fruits contains valuable constituents that have beneficial potential against some pneumonia pathogens.