Spent catalysts represent a large amount of refinery solid waste. In particular, hydro-processing catalysts contain base valuable metals, such as nickel, vanadium and molybdenum and, for their toxic component, these wastes have been classified as hazardous by the Environmental Protection Agency in the USA. The development of an innovative eco-sustainable process for the valorisation of such wastes would undoubtedly give significant advantages also taking into account primary resources preservation. This paper deals with bioleaching of metals from hazardous spent hydro-processing catalyst by means of iron/sulphur oxidizing bacteria. The exhaust catalyst was rich in nickel (45 mg/g), vanadium (44 mg/g) and molybdenum (94 mg/g). Before bioleaching, the solid was washed by means of a mixture of Tween 80 and ethyl alcohol, for hydrocarbon removal. The effects of elemental sulphur, ferrous iron and actions contrasting a possible metal toxicity (either the presence of powdered activated charcoal or the simulation of a cross current process by means of filtration stages in series) was investigated. Ferrous iron resulted to be essential for metal extraction and for bacteria adaptation. Nickel and vanadium were successfully bioleached in the presence of iron, reaching extraction yields of 83% and 90%, respectively; on the other hand extractions around 50% for nickel and vanadium were observed both in biological systems in the absence of iron and in the chemical controls with iron. As concerns molybdenum, the highest extraction yields experimentally observed was about 50%, after 26 days bioleaching in the presence of iron, while a maximum extraction of 25 % was observed in the other treatments. In conclusion, a bio-oxidative attack with iron could successfully extract nickel, vanadium and partially molybdenum. Further actions aimed at contrasting a possible metal toxicity resulted not to be effective.