Abstract <p>Extremophilic autotrophic bacteria <i>Acidithiobacillus ferrooxidans</i> are well known for their extremely acidophilic properties and wide application in biomining and metal bioleaching. Selection of <i>A. ferrooxidans</i> industrial strains by their oxidative activity commonly requires some long-term experiments. In contrast to these classical schemes, the presented research compared the strains responses to different substrates by the activity of bacterial respiration. The oxygen consumption by immobilized <i>A. ferrooxidans</i> biomass was measured using a Clark-type electrode. Amperometric express analyses showed that <i>A. ferrooxidans</i> VKM B-3655 isolated from arsenopyrite ore and VKM B-3906 isolated from iron-nickel silicate ore showed different preferences during oxidation of ferrous iron or formate, or their mixture. Simultaneous presence of two alternative substrates, instead of one, decreased the overall respiratory activity of the biomass. In this case, the combined use of formate and ferrous iron meant competition between chemolithoautotrophic and chemoorganoautotrophic processes in the bacteria. Both studied <i>A. ferrooxidans</i> strains also showed some stress reaction for added arsenite or arsenate which were tested as the most typical toxicants known for arsenopyrite ores. In general, the presented amperometric method provides an analysis of bacterial oxygen consumption as a specific respiratory response to any added substrate in extremophilic <i>A. ferrooxidans</i>.</p>