AbstractMicroorganisms are considered to play an important role in the formation of manganese oxide in natural environments. At Yunotaki Fall in north Japan, manganese‐oxidizing bacteria were previously assumed to have oxidized manganese to precipitate birnessite, which relied on oxygen released from algae (i.e., indirect oxidation sensu lato). However, it remained unclear whether larger‐scale manganese oxide precipitation was actually occurring under light conditions. This study therefore evaluated the contribution of indirect oxidation using microelectrodes to analyze local water chemistry, in addition to bulk water chemistry and DNA analyses. With the downward flow of hot spring water, pH increases because of CO2 degassing and causes the saturation index of birnessite to increase downstream. Manganese concentrations in the hot spring water decrease downstream even if it was undersaturated in birnessite, suggesting the dominance of manganese oxide deposition by microorganisms. Indeed, the deposit surface was covered by thick microbial mats, and microscopic observations and DNA analysis of the mats indicate the presence of cyanobacteria, eukaryotic algae (green algae), and manganese‐oxidizing bacteria. Microelectrode measurements at the surface of a deposit covered with eukaryotic algae showed that pH and O2 concentration profiles are characteristic of oxygenic photosynthesis and aerobic respiration. On the other hand, the light and dark conditions show nearly identical Mn(II) concentration profiles decreasing within the algal mats. These results demonstrate that low bulk pH values in the hot spring water hindered indirect oxidation despite the occurrence of active oxygenic photosynthesis and that direct oxidation by manganese‐oxidizing bacteria is considered to dominate in the investigated sample.