Nonylphenol polyethoxylate (NPnEO) surfactants, ubiquitous contaminants in sewage processing facilities, continue to receive attention due to their recalcitrance and diverse toxicity concerns. How the addition of nonylphenol diethoxylate (NP2EO) and its biodegradation products affect microbial community structure in anaerobic semi-continuous digesters was investigated by molecular techniques. Fluorescence in situ hybridization (FISH) and quantitative PCR (qPCR) were used in parallel to monitor total Bacteria, four classes of Proteobacteria, total Archaea, and two genera of acetoclastic methanogens (Methanosarcina and Methanosaeta). Beta- and Gammaproteobacteria were the dominant phyla and the relative abundances of both groups were roughly doubled following NP2EO addition. While Methanosaeta was dominant over Methanosarcina throughout, NP2EO addition and accumulation of NP nearly doubled the relative abundance of Methanosarcina. While digester performance was not affected, this study revealed that relative abundances of the key players in anaerobic digestion were altered by the addition of NP2EO.