The Kitora tumulus, which is thought to have been built around the late seventh to early eighth century, has beautiful mural paintings which were executed directly on a very thin layer of plaster in the stone chamber. When the paintings were found, the plaster was partly detached from the stone wall and the murals were therefore considered to be likely to fall off the wall with a casual touch or stimulation. Therefore, it was decided to detach the mural paintings and to store them flat and under controlled environmental conditions. This was initiated immediately after the excavation of the tumulus in 2004. However, fungal growth and biofilm development were observed within the stone chamber during the relocation work. In 2005, small holes containing black substances were observed on certain areas of the ceiling plaster, and following investigation an acetic acid bacterium, Gluconacetobacter sp., was isolated from the black substances. The bacterium was also isolated from the ceiling, floor, and east wall in the stone chamber in 2008 after the relocation of most of the paintings had been completed. These bacteria were shown to decompose calcium carbonate (CaCO3), one of the primary components of the plaster, and to produce organic acids such as acetic acid. Additionally, they were observed to decrease the pH of the culture media significantly in the presence of ethanol and glucose. This is the first example of the characterization of acetic acid bacteria isolated from decayed plaster paintings, and it is likely that microbes such as these bacteria have been involved in the deterioration of the plaster. Chemicals to treat microbes in the Kitora tumulus during the relocation work were selected on the basis of their antimicrobial efficacy, low potential to cause adverse effects on the paintings, and low level of toxicity to humans, depending on the condition of the plaster or stone in each area. However, some chemicals, especially ethanol, may act as a carbon source, which could encourage the growth of microbes and thereby the production of acids by the microbes when diluted to a low concentration or in a degraded state. Moreover, prior contamination by other microbial species in the form of a biofilm could also encourage the growth of the acetic acid bacteria by providing low-molecular-weight organic materials as a nutrient source.