Abstract Four vanadyl complexes (1–4) based on methyl, nitro, chloro, and fluoro-substituted aldimine ligands, respectively, have been designed, and the structures and geometries are optimized through DFT studies. The interactions of the complexes with DNA, as per UV absorption titration, indicate that complexes 1–3 show a hyperchromic shift and complex 4 shows a hypochromic shift, implying groove and intercalation modes of binding, respectively. The highest binding constant (Kb) value, 9.84 × 105 M–1, is obtained for the methyl-substituted complex 1. The competitive binding of the complexes (1–4), through a fluorescence assay, shows the highest Stern–Volmer constant (KSV) value of 7.81 × 103 M–1 for methyl-substituted complex 1. The interaction of the complexes with BSA, as determined by the tryptophan emission quenching method, indicates a dose-dependent quenching of the intrinsic fluorescence of BSA with increasing complex concentration. All complexes exhibit moderate radical scavenging activity compared to the standard, as per antioxidant studies. Furthermore, the predictions of molecular docking studies with both DNA and BSA support the experimental observations. The complexes cleave pBR322 DNA based on gel electrophoretic studies. The cytotoxicity study, against MCF-7 cancer cells, implies that nitro-substituted complex 2 has a higher cytotoxicity than other complexes.