A series of new (tricarbonyl)rhenium(I) complexes were synthesized using chiral bidentate ligands (+)/(-)-iminopyridines (LR/LS). The reaction yielded a mixture of mononuclear Re(I) diastereoisomers, formulated as fac-[Br(CO)3Re(S/R)L(S/R)]. Each single diastereoisomer was isolated and fully characterized. X-ray crystallography and circular dichroism spectra verified their enantiomeric nature. The cytotoxicity of each complex was evaluated against six cancer cell lines. The effect of the two complexes on viability, proliferation, and migration was analyzed on glioblastoma cell lines (U251 and LN229). Changes in the expression of histones, apoptotic, and key signaling proteins, as well as alterations in DNA structure, were also observed. These experiments showed that the chirality associated with both metal and ligand has a strong influence on cytotoxicity.