Despite its negative redox potential, nitroxyl (HNO) can trigger reactions of oxidation. Mechanistically, these reactions were suggested to occur with the intermediate formation of either hydroxyl radical (·OH) or peroxynitrite (ONOO^–). In this work, we present further experimental evidence that HNO can generate ·OH. Sodium trioxodinitrate (Na₂N₂O₃), a commonly used donor of HNO, oxidized phenol and Me₂SO to benzene diols and ·CH₃, respectively. The oxidation of Me₂SO was O₂-independent, suggesting that this process reflected neither the intermediate formation of ONOO^– nor a redox cycling of transition metal ions that could initiate Fenton-like reactions. In solutions of phenol, Na₂N₂O₃ yielded benzene-1,2-diol and benzene-1,4-diol at a ratio of 2:1, which is consistent with the generation of free ·OH. Ethanol and Me₂SO, which are efficient scavengers of ·OH, impeded the hydroxylation of phenol. A mechanism for the hydrolysis of Na₂N₂O₃ is proposed that includes dimerization of HNO to cis-hyponitrous acid (HO-N=N-OH) with a concomitant azo-type homolytic fission of the latter to N₂ and ·OH. The HNO-dependent production of ·OH was with 1 order of magnitude higher at pH 6.0 than at pH 7.4. Hence, we hypothesized that HNO can exert selective toxicity to cells subjected to acidosis. In support of this thesis, Na₂N₂O₃ was markedly more toxic to human fibroblasts and SK-N-SH neuroblastoma cells at pH 6.2 than at pH 7.4. Scavengers of ·OH impeded the cytotoxicity of Na₂N₂O₃. These results suggest that the formation of HNO may be viewed as a toxicological event in tissues subjected to acidosis.