This paper presents a route for the treatment of MSWI fly (FA) and bottom ashes (BA) using microorganisms to critically assess whether bioleaching is within reach of effective industrial application. The leaching of metals from BA and FA was investigated in a controlled laboratory environment using a culture isolated from a natural system where the dominant strains are acidophilic bacteria, mainly Acidothiobacillus thiooxidans and Acidothiobacillus ferrooxidans. The community of microorganisms (mostly acidophilic, S- and Fe-oxidizing bacteria) was collected directly from overflows and ponds at the sediment–water interface of a natural system near a post-mining site. Pre-cultivation was done in 250 mL flasks followed by the adaptation to the different substrates (both FA and BA). The effect of different material pre-treatment and elemental sulphur concentrations were evaluated for both BA and FA, at a starting pH of 4. The bioleaching of BA and FA substrates experienced good yields of metal extraction with an optimum duration of two weeks. The results showed that more than 90% Zn, Cu, and 10% Pb are removed from FA; while 100% Cu, 80% Zn and 20% Pb are removed from BA samples. Batch experiments with regenerating ion-exchange resins did not perform well for metal recovery, but could serve as a valuable decontamination step. The techniques used here with FA and BA can be used for urban mining purposes (e.g. ashes and other meal-rich anthropogenic wastes), but also low-grade ores in the mining industry, contributing to resource recovery or decontamination agendas.