Pentachlorophenol (PCP) is used in industrial and domestic applications, including as a biocide and a wood preservative. Metabolism of PCP undergoes oxidative dechlorination, forming tetrachlorocatechol (TCC) and tetrachlorohydroquinone (TCHQ). Both sodium azide (NaN3) and TCC appear naturally in soil. None of them are cytotoxic by themselves or facilitate autooxidation. Here, we show that their combination leads to synergistic cytotoxicity (>6 log bacterial killing) to Escherichia coli. The rate of oxygen consumption in a cell-free system showed that NaN3 increases TCC oxidation by 520-fold. The synergism coefficient to cells was calculated as 96 or greater, and we have shown the formation of a new compound. It is suggested that the intermediate species, o-tetrachlorosemiquinine, and an unknown, nitrogen-centered free radical, both visualized by electron-spin resonance, are harmful species responsible for the synergistic cytotoxicity of TCC/NaN3, rather than the endproduct formed during the reaction. Desferoxamine and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide offered nearly complete protection, but through radical scavenging rather than through chelating properties. The mechanism of damage for TCC compared to its analogue, TCHQ, were investigated, and whereas the cellular damage of TCHQ/NaN3 is through a site-specific mechanism, in the case of TCC/NaN3 it is through the accumulation of the component(s) in the bacterial cell membrane, eventually leading to dysfunction, as evidenced by electron microscopy.