Shiga toxin-producing Escherichia coli (STEC) include several serotypes isolated from cases of hemorrhagic colitis and, hemolytic uremic syndrome. Although O157:H7 is the most predominant STEC serotype, more than 100 non-O157 serogroups cause diseases in humans. Some STEC carry a Locus of Enterocyte Effacement (LEE-positive); however, STEC that do not carry LEE (LEE-negative) have also been associated with illness, mainly those harbouring the Locus of Adhesion and Autoaggregation (LAA). LAA carry some genes such as hes, iha, tpsA, and agn43, related with pathogenicity. One of them is the ability to form biofilms on different environments, which can contaminate food and generate infections while protecting themselves against adverse conditions. Considering that LAA could be responsible for some adherence mechanisms, the aims of this study were to compare different serogroup of LEE-negative STEC strains in their ability to form biofilms and to evaluate the participation of some genes encoding in LAA. A total of 348 LEE-negative STEC strains was analyzed. The presence of hes, iha, tpsA and agn43 were determined by monoplex PCR. From them, 48 STEC strains belonging to serogroups O113, O130, O171, O174 and, O178 were assayed for their ability to form biofilm. The most prevalent genes detected were agn43 (72.1%) and tpsA (69.5%). The iha and hes genes were present in 63.7% and 54% of the strains, respectively. Although all STEC strains were able to form biofilm, it was found a high variability between them. The relation between the biofilm formation and the presence of each gene was not statistically significant, suggesting that biofilm formation is independent of the presence of those genes. Highlighting that there is no treatment for HUS, it is once again notable that prevention measures and control strategies to prevent biofilm formation are important factors in reducing STEC transmission.