In this study, the phytochemical investigation of Scorzonera yildirimlii and S. zorkunensis led to the isolation and identification of eight new oligosaccharide constituents (1-8). Their structures were elucidated through comprehensive spectroscopic analysis (NMR, FT-IR, and mass spectrometry) and comparison with literature data. The structure of isolated compounds were O-α-D-Glcp-(1 → 2)-O-β-D-Fruf-(3 → 1)-O-myo-inositol (1), O-α-D-Glcp-(1 → 2)-O-β-D-Fruf-(3 → 1)-O-epi-inositol (2), O-β-D-Fruf-(2 → 6)-O-α-D-Glcp-(1 → 1)-O-β-D-Glcp (3), O-β-D-Fruf-(2 → 6)-O-α-D-Glcp-(1 → 1)-O-β-D-Galp (4), O-β-D-Fruf-(2 → 6)-O-β-D-Fruf-(2 → 6)-O-α-D-Glcp-(1 → 1)-O-β-D-Glcp (5), O-β-D-Fruf-(2 → 6)-[O-β-D-Glcp-(1 → 4)]-O-α-D-Glcp-(1 → 1)-O-β-D-Glcp (6), O-β-D-Fruf-(2 → 6)-[O-β-D-Glcp-(1 → 4)]-3-OCH3-O-α-D-Glcp-(1 → 1)-O-β-D-Glcp (7), and O-β-D-Fruf-(2 → 6)-[O-β-D-Glcp-(1 → 4)]-O-α-D-Glcp-(1 → 4)-O-α-D-Glcp-(1 → 2)-O-β-D-Fruf (8). The antimicrobial activities of all the isolated compounds (1-8) were investigated against six microorganisms. Compounds 2 and 3 were effective against Mycobacterium smegmatis at minimum inhibitory concentrations (MICs) of 3.13 μg/mL and 3.44 μg/mL, respectively, compared with streptomycin (MIC = 4.0 μg/mL). The enzyme-inhibitory properties of the isolated compounds (1-8) were evaluated against tyrosinase and α-glucosidase. Among the tested compounds, 4 (39.78 ± 10.29 %) exhibited inhibitory activity against tyrosinase, while compound 2 (68.04 ± 4.68 %) exhibited inhibitory activity against α-glucosidase. In silico studies of 1-8 showed the best predicted binding affinities for the Trehalose-LpqY complex in the range of -11.088 kcal/mol to -14.297 kcal/mol, compared to streptomycin (-6.638 kcal/mol). Compounds 1-8 exhibited almost no cytotoxic activity against L-929 cells, further supporting their potential for therapeutic use.