The continuous and rapid spread of the COVID-19 pandemic has emphasized the need to seek new therapeutic and prophylactic treatments. Peptide inhibitors are a valid alternative approach for the treatment of emerging viral infections, mainly due to their low toxicity and high efficiency. Recently, two small nucleotide signatures were identified in the genome of some members of the Coronaviridae family and many other human pathogens. In this study, we investigated whether the corresponding amino acid sequences of such nucleotide sequences could have effects on the viral infection of two representative human coronaviruses: HCoV-OC43 and SARS-CoV-2. Our results showed that the synthetic peptides analyzed inhibit the infection of both coronaviruses in a dose-dependent manner by binding the RBD of the Spike protein, as suggested by molecular docking and validated by biochemical studies. The peptides tested do not provide toxicity on cultured cells or human erythrocytes and are resistant to human serum proteases, indicating that they may be very promising antiviral peptides.