In the past months, the use of the drug hydroxychloroquine has considerably increased in many countries, associated with a proposed treatment for the COVID-19 disease. Although there is no conclusive evidence about the efficacy of the drug for this purpose, surprisingly there are no conclusive studies in the literature concerning its mechanism of action inside cells, which is related to its interaction with nucleic acids. Here, we performed a robust characterization of the interaction between hydroxychloroquine and double-stranded DNA using single-molecule force spectroscopy and gel electrophoresis. Two different binding modes were identified, namely, minor groove binding for low drug concentrations and intercalation for high drug concentrations, and the sets of binding parameters were determined for each of these modes. Such results have unraveled in detail the molecular mechanism of action of the drug as a DNA ligand.