Nepeta cyrenaica Quézel & Zaffran (Lamiaceae), an endemic species of Libyan flora, is here characterized for the first time for its phytochemical composition and biological activities. Phenolic composition, antioxidant and enzyme inhibiting effects of extracts, prepared from five-week-old in vitro propagated N. cyrenaica shoots, were evaluated. Extraction was performed using dichloromethane, methanol, 96% ethanol or hot distilled water. LC-MS analysis showed that the methanol extract contained the highest amount of phenolic components, especially ferulic and rosmarinic acids (1300.73 mg/kg and 528.88 mg/kg, respectively), and epigallocatechin gallate (719.05 mg/kg). The strongest antioxidant activity was recorded for aqueous extract in DPPH assay (66.91%) and dichloromethane extract in β-carotene bleaching assay (81.06%), both tested at the concentration of 2 mg/mL. Concerning α-glucosidase inhibition, dichloromethane extract was shown to possess a higher inhibition capacity than acarbose at the concentration of 2 mg/mL (95.33% vs. 88.29%). The aqueous extract exhibited higher acetylcholinesterase inhibition than the other tested extracts, which was lower compared to the positive control, galantamine. Although methanol extract contained the highest amount of polyphenolics, dichloromethane and aqueous extracts were shown to be more suitable for the extraction of bioactive components. In conclusion, endemic N. cyrenaica could be efficiently propagated through in vitro propagation protocols as a polyphenolic-rich plant with valuable medicinal potential.