Polyphenols, obtained from natural resources, may possess important pharmacological effects. The polyphenolic profiles of the stem extracts of six Ferocactus species (sp.): F. gracilis, F. pottsii, F. herrerae, F. horridus, F. glaucescens, and F. emoryi, were measured using high-performance liquid chromatography (HPLC) with diode-array detection (DAD). Additionally, anticancer, antibacterial, and antifungal activities were examined. Results showed the presence of high to moderate amounts of polyphenols in the extracts (phenolic acids: Protocatechuic acid, 3,4-dihydroxyphenylacetic acid, caffeic acid, and vanillic acid; flavonoids: Rutoside and quercitrin). The highest amounts of 3,4-dihydroxyphenylacetic acid were found in F. glaucescens ((132.09 mg 100 g−1 dry weight (DW)), F. pottsii (75.71 mg 100 g−1 DW), and F. emoryi (69.14 mg 100 g−1 DW) while rutoside content was highest in F. glaucescens (107.66 mg 100 g−1 DW). Maximum antiproliferative activities were observed against HeLa and Jurkat cancer cells, with F. glaucescens, F. emoryi, and F. pottsii showing the highest anticancer activity. Most bacteria were sensitive to Ferocactus sp. stem extracts. Escherichia coli and Staphylococcus aureus were the most sensitive. Excellent antifungal effects were observed against Aspergillus ochraceus and A. niger. However, Penicillium funiculosum, P. ochrochloron, and Candida albicans were relatively resistant. This is the first study reporting novel sources of polyphenols in Ferocactus sp. with anticancer and antimicrobial activities.