Since the fruits of Lycium L. species (Fructus Lycii, goji berries) are promoted as a “superfood” with plenty of health benefits, there is extensive research interest in their nutritional and phytochemical composition. In the present study, the nutritional value, minerals, fatty acid composition, and bioactive compounds of L. barbarum L., red, yellow, and black goji berry (L. ruthenicum Murray.) cultivated in Serbia were investigated. Antioxidant and antimicrobial properties of their methanol extracts were assessed. Red goji berry had the highest content of fats, dietary fiber, iron, total carotenoids, and 2-O-β-d-glucopyranosyl-l-ascorbic acid (AA-2βG). The yellow goji berry extract showed the highest level of flavonoids and the most prominent antimicrobial (especially against Gram-negative bacteria) properties. The highest total phenolic content and the most potent antioxidant activity were observed for the extract of black goji berry. Therefore, all goji berries could be a valuable source of bioactive compounds in the food and pharmaceutical industry.