The aim of this study was to analyze in vitro biological activities as anti-oxidant, anti-α-amylase, anti-α-glucosidase, anti-lipase, and anti-lipoxygenase activity, relative to bioactive components (phenolic acids, flavonols, xanthophylls, carotenes, esterified carotenoids, tocopherols, tocotrienols, and fatty acids) and the basic chemical composition (sugars, organic acid, dry matter, soluble solid, pH, titratable acidity, ash, pectins, and vitamin C) of Hippophaë rhamnoides berries. Six sea buckthorn cultivars commonly grown in Poland were analyzed including Aromatnaja, Botaniczeskaja-Lubitelskaja, Józef, Luczistaja, Moskwiczka, and Podarok Sadu. Berries contained 1.34–2.87 g of sugars and 0.96–4.22 g of organic acids in 100 g fresh weight, 468.60–901.11 mg of phenolic compounds, and 46.61–508.57 mg of carotenoids in 100 g dry mass. The fatty acid profile was established: palmitic > palmitoleic > oleic and linoleic > stearic and linolenic acids. The highest anti-oxidant (34.68 mmol Trolox/100 g dry mass) and anti-α-amylase potential (IC50 = 26.83 mg/mL) was determined in Aromatnaja, anti-α-glucosidase in Botaniczeskaja-Lubitelskaja (IC50 = 41.78 mg/mL), anti-lipase in Moskwiczka and Aromatnaja (average IC50 = 4.37 mg/mL), and anti-lipoxygenase in Aromatnaja and Podarok Sadu fruits (100% inhibition). The studied sea buckthorn berries may be a raw material for the development of functional foods and nutraceutical products rich in compounds with high biological activity.