This paper reports the results of a study of biogenic Mn oxide minerals coating in a subsurface granite environment.This subterranean environment corresponds to galleries of a 16th century spring water tunnel dug throughout the granite bedrock located in Porto city (NW Portugal). Several techniques (XRD, FTIR, Raman, ICP-MS, TEM-EDS, FESEM-EDS, STEM-EDS and STXM-NEXAFS) were used to assess the mechanisms involved in the formation of manganese oxides, specifically birnessite and todorokite. These manganese oxides presented peculiar shapes, nano-dimensions, low degree of crystallinity, and high levels of some trace elements such as P, Ca, C, Al, Si, Ba and Zn. They were associated with large amounts of extracellular polymeric substances exuded by filamentous bacterial communities, which serve as nuclei for preferential precipitation of manganese oxides on the extracellular sheaths. Scientific assessment revealed that biological activity played a major role in the development of these manganese oxides.