Vincristine is a classical chemotherapeutic agent widely used for its ability to disrupt microtubule polymerization, yet additional molecular effects may contribute to its anticancer activity. G-quadruplexes (G4s), non-canonical nucleic acid structures enriched in regulatory regions of the genome and in mitochondrial DNA, have emerged as relevant modulators of cellular homeostasis. In this study, we investigated whether vincristine can influence G4 biology. Cancer cells treated with vincristine were analyzed by immunofluorescence, revealing a consistent increase in nuclear and mitochondrial G4 foci. In particular, mitochondrial G4s were significantly elevated by approximately 1.5–2.5 fold compared to untreated cells, an effect accompanied by a detectable reduction in membrane potential, indicative of impaired organelle function. In addition, biophysical analyses on representative G4-forming sequences were carried out. Proton nuclear magnetic resonance titrations showed localized chemical shift perturbations upon vincristine addition, circular dichroism confirmed preservation of G4 topology, and isothermal titration calorimetry indicated weak but enthalpically favorable interactions. Taken together, these results suggest that vincristine perturbs both the cellular G4 landscape and mitochondrial homeostasis, while also engaging G4 DNA in vitro. Although additional studies are required to establish the mechanistic details, this work provides proof-of-concept for a previously unrecognized dimension of vincristine’s anticancer action.