The development of new antiviral drugs against SARS-CoV-2 is a valuable long-term strategy to protect the global population from the COVID-19 pandemic complementary to the vaccination. Considering this, the viral main protease (M-pro) is among the most promising molecular targets in light of its importance during the viral replication cycle. The natural flavonoid quercetin 1 has been recently reported to be a potent M-pro inhibitor in vitro, and we explored the effect produced by the introduction of organoselenium functionalities in this scaffold. In particular, we report here a new synthetic method to prepare previously inaccessible C-8 seleno-quercetin derivatives. By screening a small library of flavonols and flavone derivatives, we observed that some compounds inhibit the protease activity in vitro. For the first time, we demonstrate that quercetin (1) and 8-(p-tolylselenyl)quercetin (2d) block SARS-CoV-2 replication in infected cells at non-toxic concentrations, with an IC50 of 192 mu M and 8 mu M, respectively. Based on docking experiments driven by experimental evidence, we propose a non-covalent mechanism for M-pro inhibition in which a hydrogen bond between the selenium atom and Gln189 residue in the catalytic pocket could explain the higher M-pro activity of 2d and, as a result, its better antiviral profile.