Our understanding of Escherichia coli biofilm formation in vitro is based on studies of laboratory K-12 strains grown in standard media. However, pathogenic E. coli isolates differ substantially in their genetic repertoire from E. coli K-12 and are subject to heterogeneous environmental conditions. In this study, in vitro biofilm formation of 331 nondomesticated E. coli strains isolated from healthy (n = 105) and diarrhea-afflicted children (n = 68), bacteremia patients (n = 90), and male patients with urinary tract infections (n = 68) was monitored using a variety of growth conditions and compared to in vitro biofilm formation of prototypic pathogenic and laboratory strains. Our results revealed remarkable variation among the capacities of diverse E. coli isolates to form biofilms in vitro. Notably, we could not identify an association of increased biofilm formation in vitro with a specific strain collection that represented pathogenic E. coli strains. Instead, analysis of biofilm data revealed a significant dependence on growth medium composition (P < 0.05). Poor correlation between biofilm formation in the various media suggests that diverse E. coli isolates respond very differently to changing environmental conditions. The data demonstrate that prevalence and expression of three factors known to strongly promote biofilm formation in E. coli K-12 (F-like conjugative pili, aggregative adherence fimbriae, and curli) cannot adequately account for the increased biofilm formation of nondomesticated E. coli isolates in vitro. This study highlights the complexity of genetic and environmental effectors of the biofilm phenotype within the species E. coli.