Biofilm formation may increase survival and persistence of Escherichia coli in the highly variable conditions of soil environments, though it remains unknown the extent variation in biofilm formation affects survival. We asked what genetic traits influence biofilm formation in phylogroup D E. coli isolates from surface soils, and are they associated with the soil environment? Biofilm density was analyzed and compared with soil environment characteristics. Isolates produced more biofilm per unit growth at 15°C than 37°C. Biofilm formation was greater in soil isolates than fecal isolates and in soils with moisture and higher calcium and pH levels. A GWAS analysis found variants involved in cell envelope formation and structure were associated with biofilm formed at 37°C, and stress response and iron acquisition variants were associated with biofilm formed at 15°C. Motility variants were associated with a negative effect on biofilm formed and adhesion variants associated with a positive effect.