In this study, the possible changes in microbial communities after the occurrence of an earthquake were investigated. To this end, the effects of the 2016 Gyeongju earthquake (ML 5.8) on the microbial communities in nearby aquifers were analyzed together with associated hydrochemistry data. Fourteen water samples were collected from two types of wells (alluvial and fractured rock formation); hydrogeochemical parameters, groundwater level, radon concentration, strontium isotopes, and microbiological features were monitored. The results obtained for microbial compositions and special bacteria were consistent with the hydrochemical results. Distinctive microbial communities in groundwater samples taken adjacent to the earthquake epicenter were also observed. The predominant bacteria in most samples were Pseudomonas and Flavobacterium. Radon concentrations, helium isotopes, and the helium/neon ratio showed anomalies in one well (KW8) compared to microbial composition data. Strontium data indicated that the study site may have been affected by seawater intrusion; this was corroborated by the microbiological features of archaea. The results indicate that an earthquake can change the number of species or their abundance in groundwater ecosystems. Therefore, microbial features might be a good additional indicator for analyzing the effects of earthquakes on groundwater, together with major chemical parameters such as radon, strontium, and helium. The microbiological features indicate that levels of bacteria such as Pseudomonas and Flavobacterium in the groundwater were highly correlated with the Gyeongju earthquake and the associated hydrochemical data.