This study was conducted to examine the phytoconstituent, antioxidant, enzyme inhibitory, antimicrobial, and cytotoxic activities of Alkanna orientalis (L.) Boiss. and A. verecunda Hub.-Mor., both from the Boraginaceae family. Root extracts were prepared using methanol. High-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC-ESI-Q-TOF-MS) analysis results revealed the dominance of caffeic acid derivatives in both species. Both species contained variable amounts of rosmarinic acid, dimers and trimers of caffeic acids, lithospermic acid, salvianolic acid A and C, and sagerinic acid. A. orientalis demonstrated higher total phenolic (96.18 mg gallic acid equivalent (GAE)/g) and flavonoids (6.33 mg rutin equivalent (RE)/g) contents than A. verecunda, leading to increased antiradical (DPPH = 316.74 mg trolox equivalents (TE)/g; ABTS = 491.21 mg TE/g), ions reducing (CUPRAC = 790.46 mg TE/g; FRAP = 523.24 mg TE/g), chelation (7.43 mg ethylenediaminetetraacetate equivalent (EDTAE)/g), and total antioxidant (3.17 mmol TE/g) activities. Anti-acetylcholinesterase activity was comparable in both species (2.98 and 2.92 mg galantamine equivalent (GALAE)/g, p ≥ 0.05). A. orientalis most effectively inhibited α–glucosidase (2.23 mmol acarbose equivalent (ACAE)/g) and α–amylase (0.26 mmol ACAE/g), while A. verecunda showed the highest significant inhibitory effect against butyrylcholinesterase (1.98 mg GALAE/g), anti-tyrosinase (49.75 mg KAE/g), and human carbonic anhydrase isoenzyme II (IC50 0.070 µg/mL). A. orientalis exhibited significant antibacterial activity against Staphylococcus epidermidis ATCC14990, with a minimum inhibitory concentration (MIC) of 0.008 mg/mL and a minimum bactericidal concentration (MBC) of 0.0625 mg/mL. The cytotoxic activity of methanol extracts from A. orientalis and A. verecunda was evaluated on four human cancer cell lines: gastric adenocarcinoma (AGS), ovarian adenocarcinoma (SKOV-3), colon carcinoma (HCT-116), and cervical adenocarcinoma (HeLa), using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Human fibroblasts (HFF-1) were employed as a non-cancerous control line. The findings suggest that the extracts exhibited mild cytotoxic activity on cancer cells while demonstrating no toxicity on normal fibroblasts. A more potent effect was observed for A. orientalis compared to A. verecunda extracts, particularly on gastric cancer AGS cells, where cell viability decreased to 56.84 ± 4.20% at the highest concentration of A. orientalis extract. In conclusion, this study is the first to explore the phytochemical constituents and biological activities of A. verecunda and the roots of A. orientalis. The roots of both species could serve as a novel source of bioactive molecules with potential applications in the pharmaceutical and cosmetic industries.