Sulfate- and Fe(III)-reducing, and methanogenic prokaryotes (SRP, FRP, MGP) are key players in metabolic pathways involved in anaerobic biodegradation processes. Understanding the metabolic activity of these microbes in environments can enhance microbe-mediated processes such as oil spill bioremediation and methane biogas production. In this study, anaerobic microbial activities in Deepwater Horizon oil spill-impacted salt marsh sediments, and in methanogenic coal bed production water enrichment cultures amended with trace elements (TE), were elucidated by employing an approach combining methods in molecular biology and geochemistry. In situ metabolic activity of SRP, FRP and MGP were monitored seasonally and metabolically-active communities were identified in oil-impacted sediments using quantitative real time Reverse Transcription -PCR and clone library analysis of key functional genes: Dissimilatory (bi)sulfite reductase (dsrAB), Geobactereceae-specific citrate synthase (gltA), methyl coenzyme M reductase (mcrA), and benzyl succinate synthase (bssA). In situ application of montmorillonite clay was assessed for its potential at accelerating PHC degradation by stimulating microbial activities. Levels of dsrA, gltA and bssA transcripts suggested that PHC-oxidizing SRP are more active in summer while FRP are more active in winter, indicating their activities linked to the seasonal changes of redox potential and vegetation. BssA gene expression peaked in winter, and was highest at more highly oil-impacted sites. Expression of all genes was higher in clay-amended sites. bssA transcript level and Fe(II) production were highest in clay-amended microcosm. Total petroleum hydrocarbon (TPH) levels were lower in oil and clay-amended microcosm incubation than one with oil only amendment, suggesting enhanced TPH degradation by clay amendment. Pyrosequencing analysis 16S rRNA gene in clay-amended microcosms demonstrated the highest percentage abundance of groups closely related to known anaerobic aromatic degraders. Levels of mcrA transcripts correlated with methane production rates in TE-amended coal bed production water enrichments. The findings of the present study clearly support the advantage of gene expression analyses for estimating microbial activity. To the best of our knowledge, this is the first in situ study which employs key functional gene markers as molecular proxies for metabolic activity and diversity assessments in anaerobic oil-contaminated salt marsh sediment and also elucidates clay-enhanced in situ TPH degradation.