Contamination of groundwater with vinyl chloride (VC), a known human carcinogen, is a common environmental problem at plastics manufacturing, dry cleaning, and military sites. At many sites, there is the potential to cleanup VC groundwater plumes with aerobic VC-oxidizing microorganisms (e.g., methanotrophs, etheneotrophs, and VC-assimilating bacteria). Environmental biotechnologies that reveal the presence and activity of VC-oxidizing bacteria in contaminated groundwater samples would provide valuable lines of evidence that bioremediation of VC is occurring at a site. We applied targeted shotgun mass spectrometry-based proteomic methods to ethene-enriched groundwater microcosms from a VC-contaminated site. Polypeptides from the enzymes alkene monooxygenase (EtnC) and epoxyalkane:CoM transferase (EtnE), both of which are expressed by aerobic etheneotrophs and VC-assimilating bacteria, were identified in 7 of the 14 samples analyzed. Bioinformatic analysis revealed that 2 EtnC and 5 EtnE peptides were unique to deduced EtnC and EtnE sequences from two different cultivated strains. In addition, several partial EtnE genes sequenced from microcosms matched with observed EtnE peptides. Our results have revealed broader etheneotroph functional gene diversity and demonstrate the feasibility, speed, and accuracy of applying a targeted metaproteomics approach to identifying protein biomarkers from etheneotrophs in complex environmental samples.