Herein we report for the first time the screening of several ligands in terms of their ability to bind and stabilize G-quadruplexes found in seven human Papillomavirus (HPV) genomes. Using a variety of biophysical assays, HPV G-quadruplexes were shown to possess a high degree of structural polymorphism upon ligand binding which may have an impact on transcription, replication and viral protein production. A sequence found in high-risk HPV16 genotype folds into multiple non-canonical DNA structures; it was converted into a major G-quadruplex conformation upon interaction with a well-characterized highly selective G4-ligand, PhenDC3, which may have animpact on the viral infection. Likewise, HPV57 and 58, which fold into multiple G-quadruplex structures, were found to form single stable complexes in the presence of two other G4-ligands, C8and Pyridostatin, respectively. Additionally, one of the selected compounds, the acridine derivative C8, demonstrated a significant antiviral effect in HPV18-infected organotypic raft cultures. Altogether, these results indicate that targeting HPV G-quadruplexes may be an alternative route for the development of novel antiviral therapies.