This thesis discusses the use of mass spectrometry and polymerase chain reaction (PCR), among other methods, to detect biomarkers of microorganisms in the environment. These methods can be used to detect bacteria involved in the degradation of environmental pollutants (bioremediation) or various single-celled pathogens, including those posing potential threats as bioterrorism agents. The first chapter introduces the hurdles in detecting in diverse environmental compartments in which they could be found, a select list of single-celled pathogens representing known or potential bioterrorism agents. These hurdles take the form of substances that interfere either directly or indirectly with the detection method. In the case of mass spectrometry-based detection, many of these substances (interferences) can be removed via effective sample pretreatment. Chapters 2 through 4 highlight specific methods developed to detect bioremediation or bioterrorism agents in environmental matrices. These methods are qualitative mass spectrometry, quantitative PCR, and quantitative mass spectrometry, respectively. The targeted organisms in these methods include several bioremediation agents, e.g. Pseudomonas putida F1 and Sphingomonas wittichii RW1, and bioterrorism agents, e.g. norovirus and Cryptosporidium parvum. In Chapter 2, I identify using qualitative mass spectrometry, biomarkers for three bacterial species involved in bioremediation. In Chapter 3, I report on a new quantitative PCR method suitable for monitoring of a key gene in yet another bioremediation agent, Sphingomonas wittichii RW1; furthermore, I apply this method to track the efficacy of bioremediation in bioaugmented environmental microcosms. In Chapter 4, I report on the development of new quantitative mass spectrometry methods for two organisms, S. wittichii RW1 and Cryptosporidium parvum, and evaluate two previously published methods for their applicability to the analysis of complex environmental samples. In Chapter 5, I review state-of-the-art methods for the detection of emerging biological contaminants, specifically viruses, in environmental samples. While this summary deals exclusively with viral pathogens, the advantages and remaining challenges identified are also applicable to all single-celled organisms in environmental settings. The suggestions I make at the end of this chapter are expected to be valid not only for future needs for emerging viruses but also for bacteria, eukaryotic pathogens, and prions. In general, it is advisable to continue the trend towards quantification and to standardize methods to facilitate comparison of results between studies.