Chilean hop (Humulus lupulus L.) ecotypes are an under-explored resource with high bioactive potential, offering promising applications in food preservation and health promotion. This study aimed to characterize the chemical composition and evaluate the antioxidant, antimicrobial, and cytotoxic properties of methanolic extracts from three native ecotypes—Ranco, La Unión, and Valdivia—to identify their potential as sources of multifunctional bioactive compounds. Each ecotype exhibited a distinct composition of bioactive compounds; Valdivia stood out for its pronounced levels of α- and β-acids and xanthohumol. Antioxidant capacity, assessed by DPPH, FRAP, and ABTS, was strong across extracts, with Valdivia showing the highest values in all the tests carried out. The extracts inhibited multidrug-resistant clinical isolates, notably Enterococcus faecalis and Pseudomonas aeruginosa, and showed dose-dependent cytotoxic effects in H1299 and MCF-7 cell lines, with the La Unión extract particularly active against H1299. Overall, these findings position Chilean hop ecotypes as promising sources of natural antioxidants and antimicrobial agents for functional food and nutraceutical applications.