Non-canonical four-stranded G-quadruplex (G4) structures form through self-recognition of guanines into stacked tetrads within chromatin DNA. Considerable evidences exist for G4s formation in vitro and have linked G4 formation with key biological processes ranging from transcription and translation to genome instability and cancer. However, G4s have not been exploited for colorectal cancer (CRC) treatment before. Our aim was to assess G4s as therapeutic targets in CRC tumorigenesis and characterize the role of G4-mediated genomic instability in CRC. We measured the presence of G4s and DNA damage at G0/G1 and S phase of cell cycle by immunofluorescence and Fiji quantification (N > 200) using antibodies against G4s (termed BG4) and gH2AX respectively in a cellular progression model of CRC. In addition, antitumoral activity of well-known G4-ligands was assessed by Resazurin method. Genomic instability driven by these G4-ligands was also evaluated by gH2AX immunofluorescence. Non-tumoral epithelial cells showed the lowest G4 and gH2AX levels, which significantly increased along CRC progression, suggesting a role in CRC tumorigenesis. Both G4s and gH2AX levels were positively correlated and the typical genome instability in CRC was partially explained by G4s presence. G4-ligands showed cytotoxic activity and significantly induced DNA damage. Subsequent studies are required to further evaluate the therapeutic potential of G4s and associated genomic instability in CRC.