The development of molecular microbial ecology in the 1990s has allowed scientists to realize that microbial populations in the natural environment are much more diverse than microorganisms so far isolated in the laboratory. This finding has exerted a significant impact on environmental biotechnology, since knowledge in this field has been largely dependent on studies with pollutant-degrading bacteria isolated by conventional culture methods. Researchers have thus started to use molecular ecological methods to analyze microbial populations relevant to pollutant degradation in the environment (called environmentally relevant microorganisms, ERMs), although further effort is needed to gain practical benefits from these studies. This review highlights the utility and limitations of molecular ecological methods for understanding and advancing environmental biotechnology processes. The importance of the combined use of molecular ecological and physiological methods for identifying ERMs is stressed.