Shiga toxin-producing Escherichia coli (STEC) strains are often found in food and cause human infections. Although STEC O157:H7 is most often responsible for human disease, various non-O157 subtypes have caused individual human infections or outbreaks. The importance of STEC serogroup typing is decreasing while detection of virulence gene patterns has become more relevant. Whole genome sequencing (WGS) reveals the entire spectrum of pathogen information, such as toxin variant, serotype, sequence type, and virulence factors. Flour has not been considered as a vector for STEC; however, this product has been associated with several STEC outbreaks in the last decade. Flour is a natural product, and milling does not include a germ-reducing step. Flour is rarely eaten raw, but the risks associated with the consumption of unbaked dough are probably underestimated. The aim of this study was to determine the prevalence of STEC in flour samples (n = 93) collected from Swiss markets and to fully characterize the isolates by PCR assay and WGS. The prevalence of STEC in these flour samples was 10.8% as indicated by PCR, and a total of 10 STEC strains were isolated (two flour samples were positive for two STEC subtypes). We found one stx2-positve STEC isolate belonging to the classic serogroups frequently associated with outbreaks that could potentially cause severe disease. However, we also found several other common or less common STEC subtypes with diverse virulence patterns. Our results reveal the benefits of WGS as a characterization tool and that flour is a potentially and probably underestimated source for STEC infections in humans