Asphalt concrete linings (ACLs) are frequently used in pumped-storage hydropower schemes with frequently oscillating reservoir water levels. ACLs can be damaged during operation, which shortens their service life. One type of damage is the formation of blisters. In this study, the bacterial diversity in damaged and undamaged portions of the ACL of an upper water reservoir was evaluated. Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) was employed for the identification of the bacterial strains. No bacteria were detected growing inside the undamaged dense layer. Low bacterial diversity (2 and 4 morphotypes) was found in the upper part that has no contact with the reservoir water and in the lowest part that is permanently under water, respectively. More complex bacterial diversity (16 morphotypes covered by 2 different genera and 4 species) was found on the mastic coating of the ACL, which was alternately under and above the water. Very complex bacterial compositions (53 morphotypes covered by at least 13 different species from 6 genera) were found in samples taken from open blisters. Bacterial genera that are known to utilize asphalt products as a source of nutrition were found in the open blisters. The results reflect the importance of bacterial diversity in blisters and their possible role in ACL degradation.