Based on the analysis of 16S rRNA gene metabarcoding, here we report the shift in the microbial community structure along a horizontal oxygen gradient (0.40-6.06 mg L−1) in a volcanic influenced acid rock drainage (VARD) environment, known as San Cayetano (Cartago, Costa Rica; pH =2.94-3.06, sulfate ~0.87-1.19 g L−1, iron ~35-61 mg L−1). This VARD is dominated by microorganisms involved in the geochemical cycling of iron, sulfur and nitrogen; however, the identity of the species changes with the oxygen gradient along the river course. The anoxic spring of San Cayetano is dominated by a putative anaerobic sulfate-reducing Deltaproteobacterium as well as sulfur-oxidizing bacteria (such as Acidithiobacillus or Sulfobacillus), which favor the process of dissolution of sulfide minerals and oxidation of H2S. In oxic conditions, aerobic iron-oxidizers (Leptospirillum, Acidithrix, Ferritrophicum, Ferrovum) and heterotrophic bacteria (Burkholderiaceae Betaproteobacterium, Trichococcus, Acidocella) were identified among others. Thermoplasmatales archaea closely related to environmental phylotypes found in other ARD/AMD niches were also found throughout the entire ecosystem. This work describes the changes in bacterial diversity, and possible metabolic activities occurring along a horizontal oxygen gradient in a volcanic influenced acid rock drainage system.