Goji berries are undoubtedly a source of potentially bioactive compounds but their phytochemical profile can vary depending on their geographical origin, cultivar, and/or industrial processing. A rapid and cheap extraction of the polyphenolic fraction from Lycium barbarum cultivars, applied after homogenization treatments, was combined with high-performance liquid chromatography (HPLC) analyses based on two different methods. The obtained hydroalcoholic extracts, containing interesting secondary metabolites (gallic acid, chlorogenic acid, catechin, sinapinic acid, rutin, and carvacrol), were also submitted to a wide biological screening. The total phenolic and flavonoid contents, the antioxidant capacity using three antioxidant assays, tyrosinase inhibition, and anti-Candida activity were evaluated in order to correlate the impact of the homogenization treatment, geographical origin, and cultivar type on the polyphenolic and flavonoid amount, and consequently the bioactivity. The rutin amount, considered as a quality marker for goji berries according to European Pharmacopeia, varied from ≈200 to ≈400 µg/g among the tested samples, showing important differences observed in relation to the influence of the evaluated parameters.