Peroxynitrite anion is a reactive and short-lived species and its formation in vivo has been implicated in several human diseases. In view of the potential usefulness of compounds that can protect against peroxynitrite or their reactive intermediates, a study focused on flavonoid compounds was carried out. Since the reactivity of peroxynitrite may be modified by Co2/HCO3-, which is an important plasma buffer, the protection of flavonoids against peroxynitrite was evaluated by their ability to inhibit the peroxynitrite-mediated dihydrorhodamine (DHR123) oxidation with or without physiological concentrations of bicarbonate. Flavonoids from different classes were studied to elucidate which structural features are required for an effective protection. The most efficient flavonoids on protecting DHR123 against oxidation by peroxynitrite have their ability diminished in the presence of bicarbonate, but they maintain the hierarchy established in the absence of bicarbonate. The flavones are the most effective flavonoids and their effects depend mainly on the number of hydroxyl groups. These must include either a catechol group in the B-ring or a hydroxyl group at the 3-position. This work also included some isoflavones, flavanones and a flavanol, which enable us to conclude about the importance of another structural feature: the 2,3-double bond. These results indicate that the ability of flavonoids to protect against peroxynitrite depends on some structural features, also important to scavenge oxygen free radicals and to chelate metal ions. The most efficient flavonoids are effective at low concentrations with IC50 of the same magnitude as Ebselen, a selenocompound that has been reported to be excellent at protecting against peroxynitrite. Their effectiveness at low concentrations is an important aspect to take into account when characterizing a compound as an antioxidant with biological interest.