Acidithiobacillus is a genus of the Acidithiobacillia in the phylum "Pseudomonadota". This genus includes ten species of acidophilic microorganisms capable of sulfur and/or iron oxidation: Acidithiobacillus albertensis, Acidithiobacillus caldus, Acidithiobacillus cuprithermicus, Acidithiobacillus ferrianus, Acidithiobacillus ferridurans, Acidithiobacillus ferriphilus, Acidithiobacillus ferrivorans, Acidithiobacillus ferrooxidans, Acidithiobacillus sulfuriphilus, and Acidithiobacillus thiooxidans. A. ferooxidans is the most widely studied of the genus, but A. caldus and A. thiooxidans are also significant in research. Like all "Pseudomonadota", Acidithiobacillus spp. are Gram-negative and non-spore forming. They also play a significant role in the generation of acid mine drainage; a major global environmental challenge within the mining industry. Some species of Acidithiobacillus are utilized in bioleaching and biomining. A portion of the genes that support the survival of these bacteria in acidic environments are presumed to have been obtained by horizontal gene transfer.

Acidithiobacillus are chemolithoautotrophs that can occur as acidophilic, mesophilic, or mesothermophilic. Acidithiobacillus caldus can also grow mixotrophically. Currently, the genus comprises ten species which are capable of obtaining energy by oxidizing sulfur compounds, with certain species also utilizing both ferrous and ferric iron. Some species have also evolved to use hydrogen and nitrogen from the environment. They assimilate carbon from carbon dioxide using the transaldolase variant of the Calvin-Benson-Bassham cycle. The genus comprises motile, rod-shaped cells that can be isolated from low pH environments including low pH microenvironments on otherwise neutral mineral grains.

The order Acidithiobacillales (i.e. Thermithiobacillus) were formerly members of the Gammaproteobacteria, with considerable debate regarding their position and that they could also fall within the Betaproteobacteria, but the situation was resolved by whole-genome alignment studies and both genera have been reclassified to the new class Acidithiobacillia.

Some members of this genus were classified as Thiobacillus spp., before they were reclassified in 2000.

Species within Acidothiobacillus are used in the biohydrometallurgy industry in methods called bioleaching and biomining, whereby metals are extracted from their ores through bacterial oxidation. Biomining uses radioactive waste as an ore with the bacteria to obtain gold, platinum, polonium, radon, radium, uranium, neptunium, americium, nickel, manganese, bromine, mercury, and their isotopes.

Acidithiobacillus ferrooxidans has emerged as an economically significant bacterium in the field of biohydrometallurgy, in the leaching of sulfide ores since its discovery in 1950 by Colmer, Temple and Hinkle. The discovery of A. ferrooxidans led to the development of biohydrometallurgy, which deals with all aspects of microbial mediated extraction of metals from minerals or solid wastes and acid mine drainage.^{[full citation needed]} A. ferrooxidans has been proven as a potent leaching organism, for dissolution of metals from low-grade sulfide ores. Recently, the attention has been focused upon the treatment of mineral concentrates, as well as complex sulfide ores using batch or continuous-flow reactors.

Acidithiobacillus ferrooxidans is commonly found in acid mine drainage and mine tailings. The oxidation of ferrous iron and reduced sulfur oxyanions, metal sulfides and elementary sulfur results in the production of ferric sulfate in sulfuric acid, this in turn causes the solubilization of metals and other compounds. As a result, A. ferrooxidans may be of interest for bioremediation processes. Acidithiobacillus is also commonly abundant upon inner surfaces of sewers in areas exhibiting corrosion; genetic sequencing identifies Acidothiobacillus thiooxidans as the usual species present, although it is occasionally absent from such locations.

Acidithiobacillus spp. occur as single cells or occasionally in pairs or chains, depending on growth conditions. Highly motile species have been described, as well as nonmotile ones. Motile strains have a single flagellum with the exception of A. albertensis, which has a tuft of polar flagella and a glycocalyx. Nitrogen fixation also is an important ecological function carried out by some species in this genus, as is growth using molecular hydrogen as a source of energy - neither property is found in every species. Ferric iron can be used by some species as a terminal electron acceptor.