Heterotrophic biofilm growth is typical in streams receiving airport deicer runoff; however detailed studies of biofilms in this setting are rare. Sample collection for this study was done during and surrounding two deicer seasons (i.e., 2009-2010 and 2010-2011), with additional sample collection occurring in 2014. Field surveys were used to document biofilm prevalence and characteristics, as well as stream characteristics. Collected biofilm samples were analyzed via microscopy, quantitative real-time polymerase chain reaction (qPCR), microarray, gas chromatography (of a cultured isolate), as well as via sequencing (Sanger and massively parallel). Sequence data are provided elsewhere (as described in the larger citation). Water-quality and quantity data were also collected in an attempt to assess relevant environmental conditions. Water quality data included grab samples collected at the time of biofilm field surveys as well as flow-weighted composite samples that were collected throughout the study period at nearby stream gages. Continuous streamflow and temperature were also collected at these gaged sites. Additional sensors were deployed at non-gaged sites to measure water temperature at these sites. Continuous temperature data were used to calculate antecedent characteristics for various time windows. Dye tracer results allowed for the determination of flow-based times of travel between sites. Taken together with flow composite sample data (at the downstream gage closest to the airport), this allowed for the calculation of estimated water quality characteristics at downstream sites as well as the subsequent calculation of antecedent water quality characteristics at all downstream sites. Regression models were run to investigate the influence of environmental factors on biofilm volume and dissolved oxygen concentration. Models were developed by ordinary least-squares regression using the R project for statistical computing with core functionality. Predictor variables for these models are included in the data file and input files provided. These include biofilm volumes, dissolved oxygen, COD concentration, water temperature, and monitoring site designation.