This study reported the effectiveness of the newly synthesized Schiff base, 2-{(E)-[(2,4-dihydroxyphenyl)methylidene]amino}-3-methyl-butanoic acid (LVDHBSB) from chiral L-valine and 2,4-dihydroxy benzaldehyde and characterized with UV, FTIR and 1H NMR techniques. The QSAR, DFT and docking properties in silico studies of the novel Schiff base were also performed. From the results, this work conducted the brine shrimp toxicity, antimicrobial, antidiabetic and antioxidant activities to confirm the biological efficacy of novel L-valine based Schiff base compound. The docking analysis against seven proteins showed the binding scores of -3 to -9 kcal/mol. The Schiff base is supported regardless of the docking values (from -30.49 to -49.86 kcal/mol) in the offline workbench-3. Experimental biological tests exposed good results in brine shrimp lethal assay (LC50 = 208.45 µg/mL), antibacterial activity (ZI = 9-13 mm/150 µg/mL, MIC = 100-150 µg/mL), antidiabetic activity (IC50 = 504.54 µg/mL) and antioxidant (IC50 = 1773 µg/mL) respectively. The compound exhibited good antimicrobial activity within the toxicity limit and the other results are higher than LC50. © 2025 Elsevier B.V., All rights reserved.