© Springer International Publishing Switzerland 2015. Cold seeps are unique ecosystems that are generally characterized by high salinity and reducing solutions. Seepage fluid, the major water influx of this system, contains hypersaline water, sediment pore water, and other components. The Thuwal cold seeps were recently discovered on the continental margin of the Red Sea. Using 16S rRNA gene pyro-sequencing technology, microbial communities were investigated by comparing samples collected in 2011 and 2013. The results revealed differences in the microbial communities between the two sampling times. In particular, a significantly higher abundance of Marine Group I (MGI) Thaumarchaeota was coupled with lower salinity in 2013. In the brine pool, the dominance of Desulfobacterales in 2011 was supplanted byMGI Thaumarchaeota in 2013, perhaps due to a reduced supply of hydrogen sulfide from the seepage fluid. Collectively, this study revealed a difference in water components in this ecosystem between two sampling times. The results indicated that the seawater in this cold seep displayed a greater number of characteristics of normal seawater in 2013 than in 2011, which might represent the dominant driving force for changes in microbial community structures. This is the first study to provide a temporal comparison of the microbial biodiversity of a cold seep ecosystem in the Red Sea.