Shiga toxin-producing Escherichia coli (STEC) strains are important foodborne pathogens. Among these, E. coli O157:H7 is the most frequently isolated STEC serotype responsible for foodborne diseases. However, the non-O157 serotypes have been associated with serious outbreaks and sporadic diseases as well. It has been shown that various STEC serotypes are capable of forming biofilms on different food or food contact surfaces that, when detached, may lead to cross-contamination. Bacterial cells at biofilm stage also are more tolerant to sanitizers compared with their planktonic counterparts, which makes STEC biofilms a serious food safety concern. In the present study, we evaluated the potency of biofilm formation by a variety of STEC strains from serotypes O157:H7, O26:H11, and O111:H8; we also compared biofilm tolerance with two types of common sanitizers, a quaternary ammonium chloride-based sanitizer and chlorine. Our results demonstrated that biofilm formation by various STEC serotypes on a polystyrene surface was highly strain-dependent, whereas the two non-O157 serotypes showed a higher potency of pellicle formation at air-liquid interfaces on a glass surface compared with serotype O157:H7. Significant reductions of viable biofilm cells were achieved with sanitizer treatments. STEC biofilm tolerance to sanitization was strain-dependent regardless of the serotypes. Curli expression appeared to play a critical role in STEC biofilm formation and tolerance to sanitizers. Our data indicated that multiple factors, including bacterial serotype and strain, surface materials, and other environmental conditions, could significantly affect STEC biofilm formation. The high potential for biofilm formation by various STEC serotypes, especially the strong potency of pellicle formation by the curli-positive non-O157 strains with high sanitization tolerance, might contribute to bacterial colonization on food contact surfaces, which may result in downstream product contamination.