A new N3-tridentate imine ligand, 2,6-diacetylpyridinediphenylhydrazone (DAPH) (L), and its Pd(II), V(IV)O, and Ag(I) complexes were synthesized and characterized via elemental analysis, FT-IR, NMR, molar conductance, and magnetic susceptibility measurements. The obtained data confirmed geometrical structures of Pd(II), Ag(I), and V(IV)O complexes as square planar, tetrahedral, and distorted square pyramidal, respectively. Minimum inhibitory concentrations (MIC) were used to probe in vitro antimicrobial activity of DAPH ligand and metal complexes using three different bacteria. Results revealed that PdDAPH complex exhibited the highest toxicity and lowest MIC (1.25 µg/mL) toward Escherichia coli. Moreover, cytotoxic activity of the prepared complexes was evaluated via three human cancer cell lines, hepatic carcinoma (HepG2), breast carcinoma (MCF-7), and colon carcinoma (HCT-116). Among all tested complexes, PdDAPH caused a significant loss of cell viability in less time and lower dose than the reference drug vinblastine. Antioxidant activity was also measured for all complexes and compared to vitamin C. Probing the interaction of the prepared metal-DAPH chelates with calf-thymus DNA showed the Pd(II) complex displayed the strongest interaction, with a binding constant of 6.02 kcal mol−1. Molecular docking was also investigated on all complexes, with PdDAPH being the most promising compound due to its facile hydrophobic interactions with the active pocket of glucose transporter (GLUT1) enzyme. Overall, the combined findings of this work clearly demonstrate that these new compounds hold promise as efficient antibiotic and anticancer agents.