The development of unconventional oil and gas (UOG) resources results in production of large volumes of wastewater containing a complex mixture of hydraulic fracturing chemical additives and components from the formation. Release of these wastewaters into the environment poses potential risks that are poorly understood. Microbial communities in stream sediments form the base of the food chain, and may serve as sentinels for changes in stream health. Iron reducing organisms have been shown to play a role in the biodegradation of a wide range of organic compounds, and to evaluate their response to UOG wastewater, we enriched anaerobic microbial communities from sediments collected upstream (background) and downstream (impacted) of an UOG wastewater injection disposal facility in the presence of hydraulic fracturing fluid (HFF) additives: guar gum, ethylene glycol, and two biocides, DBNPA and Bronopol. Iron reduction was significantly inhibited early in the incubations with the addition of biocides, whereas amendment with guar gum and ethylene glycol stimulated iron reduction relative to unamended controls. Changes in the microbial community structure were observed across all treatments, indicating the potential for even small amounts of UOG wastewater components to influence natural microbial processes. Microbial community structure differed between enrichments with background and impacted sediments, suggesting that impacted sediments may have been pre-conditioned by exposure to wastewater. These experiments demonstrated the potential for biocides to significantly decrease iron reduction rates immediately following a spill, and demonstrated how microbial communities previously exposed to UOG wastewater may be more resilient to additional spills.Importance:Organic components of UOG wastewater can alter microbial communities and biogeochemical processes, which could alter the rates of essential natural attenuation processes. These findings provide new insights into microbial responses following a release of UOG wastewaters, and are critical for identifying strategies for remediation and natural attenuation of impacted environments.