Studies show that microorganisms resistant to numerous antibiotics spread infections, lengthen hospital stays, and increase fatalities. Amplification factors increase the demand for innovative drug-resistant disease-fighting chemicals. This research synthesised the chiral L-phenyl alanine condensed with a 2, 4-dihydroxy benzaldehyde Schiff base for biological efficacy investigations such as antimicrobial, antidiabetic, DPPH free radical, MTT assay against HeLa cells and hemolysis studies after the characterization. Derived Schiff base showed good inhibition against K. pneumoniae (G-ve), Staphylococcus aureus (G + ve), and Candida albicans (Fungus) at a 50 mu g/mL concentration. Minimum inhibitory concentration report exists in between 35 ppm and 45 ppm. It also exhibited IC50 concentrations between 138 and 265 mu g/mL in the remaining biological studies. This study uses molecular docking with the help of mcule, the CLC drug discovery work bench and visual studio applications to justify the derived compound biological activity and used work related proteins such as 1HSK, 1XCW, 3K0K, 3FDN and 3GEY for docking study. The compound showed a good binding affinity score against the targets with negative values. This study covers drug-likeness using Spartan-14 HOMO-LUMO energy levels and Swiss ADME to determine the molecular properties of the chemical structure. Theoretical outcomes are compared with commercial drugs and observed nearby results. Both theoretical outcomes supported the experimental biological activities and were good coincidence.Communicated by Ramaswamy H. Sarma Biological and theoretical investigations on 2, 4- Dihydroxy benzaldehyde condensed L-Phenyl alanine Schiff base