Glucosinolates are abundant in Brassicaceae vegetables, and they are degraded into various organic breakdown products (BPs) (R-CN, -NCS and -SCN) by myrosinase when plant tissues are damaged. This study was designed to investigate whether these BPs could be broken further into goitrogenic thiocyanate anions (SCN(-) ) metabolically and/or spontaneously. Ten glucosinolates were chosen for this study based on the various structures of their side chains. SCN(-) and cyanide anions (CN(-) ) liberated from the BPs of these glucosinolates were quantified after incubation with human liver S9 and rhodanese. Upon treatment with metabolic enzymes, CN(-) was produced from all organic thiocyanates, aliphatic and benzyl nitriles, then a substantial amount of produced CN(-) was further metabolized to SCN(-) by rhodanese. All organic thiocyanates and allyl isothiocyanate were metabolized to produce SCN(-), without involving CN(-) production. Spontaneous degradation to SCN(-) in an aqueous environment was observed only in 4-(methylthio)butyl thiocyanate, though the enzymatic reaction rate exceeded the spontaneous one. Among these BPs, the major source of SCN(-) was organic thiocyanates. The results show that some organic nitriles, organic thiocyanates and allyl isothiocyanate may be regarded as potential sources of SCN(-) through metabolism when people ingest glucosinolate-containing vegetables.