A large spill of wastewater from oil and gas operations was discovered adjacent to Blacktail Creek near Williston, North Dakota in January 2015. To determine the effects of this spill on streambed microbial communities over time, bed sediment samples were taken from Blacktail Creek upstream, adjacent to, and at several locations downstream from the spill site. Blacktail Creek is a tributary of the Little Muddy River, and additional samples were taken upstream and downstream from the confluence of Blacktail Creek and the Little Muddy River. Samples were collected in February 2015, June 2015, June 2016, and June 2017. DNA was extracted from these sediments, and sequencing of the 16S ribosomal RNA gene was performed to enable analysis of the microbial community structure. Raw sequence data was processed, and taxonomy was assigned based on the Silva 132 database (Yilmaz et al, 2014) using the MOTHUR software package (Schloss et al, 2009). Raw sequence data are available from GenBank at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA666160.