Common hop (Humulus lupulus L.) has significant health-promoting properties. Hop cones contain resins, essential oils, proteins, polyphenols, lipids, waxes, and cellulose. Hop extracts include bioactive compounds such as polyphenolic compounds (phenolic acids, and flavonols), and chlorophylls. The aim of this study was to compare the pro-health potential of hop cone extracts obtained from three cultivars (Magnum, Lubelski, and Marynka). The results showed that the cones of Magnum cultivar demonstrated the highest biological activity. The sum of phenolic acids and flavonols in ethanol extract was the highest for this variety and was equal 4903.5 µg/g dw. Ethanol extracts of Magnum cultivars showed the highest degree of iron ion chelation (55.43–88.76%) as well as the activity against 1,1-diphenyl-2-picrylhydrazyl radical (4.75 mmol Tx/g dw). Hop cone extracts as cholinesterase inhibitors showed high potential for aqueous variants. In terms of antimicrobial activity, all investigated extracts demonstrated strong inhibition against Staphylococcus aureus and Staphylococcus epidermidis, with the Magnum cultivar showing the strongest inhibition. Owing to the biofunctional features of hop cone, it can be concluded that it is an attractive raw material with pro-health potential that can be used much more widely in food technology. However, it should be noted that toxicological tests and in vitro tests must be carried out before the raw material is used in food production.