In this research, a novel L-tryptophan-vanillin Schiff base and three complexes were synthesised, characterization, and their biological activities were also determined. The physicochemical parameters of the compounds investigated revealed that, Schiff base ligand (SL) melting point = 84–85 °C, Schiff base ligand–Fe2+ complex = 245–246 °C, Schiff base ligand–Co2+complex = 271–272 °C, and Schiff base ligand–Ni2+complex > 350 °C. Molar conductivities was found to be 10,300, 5000, 17,300, and 52,900 Sm2 mol−1 for the Schiff base and the complexes, respectively. It indicates that the complexes are non-electrolytic in nature. The magnetic susceptibilities of the complexes were found to be higher than the spin-only values due to unpaired electrons (Fe2+ complex = 5.73 BM, Co2+ complex = 4.52 BM, and Ni2+ complex = 3.46 BM), suggesting octahedral geometries. Electronic absorption (max) for Fe2+ = 480, Co2+ = 520 nm, while Ni2+ shows two bands at 480 and 570 nm, which signifies the n → π* and π → π* transitions, respectively. Their crystallinity index was also assessed using the PXRD and it revealed that complexes are purely crystalline. Antimicrobial test results revealed that Co2+ and Fe2+ complexes have excellent activities against gram-negative bacteria (E. coli, F. Shigella, and S. Typhi). They are all effective against MRSA, K. Pneumoniae., and S. Pneumoniae, as well as A. niger and C. albicans. Therefore, based on the findings from the study, it was concluded that the Schiff base is a bidentate ligand, and octahedral geometries and paramagnetic were suggested for the complexes.