Shiga toxin-producing Escherichia coli (STEC) is a bacterial pathogen that cause diarrhea and severe human diseases. Its principal virulence factor are the Shiga toxins Stx1 and Stx2 which have been identified diverse subtypes considered to be responsible for severe complications of STEC infection. These toxins are encoded in temperate bacteriophages and their expression is linked to phage lithic cycle, which is regulated by late genes and the Q anti-terminator protein. The aim of this study was to characterize biologically and molecularly STEC strains encoding stx2 gene isolated from cattle feces in Colombia. We selected six STEC strains, which were evaluated its Stx production, the Stx2 subtypes, induction of the lithic cycle of bacteriophages and its late region. The results evidenced two highlighted strains with high levels of Stx production and induction of the lithic cycle, compared with the others. Likewise, the strains evaluated showed three Stx2 subtypes: Stx2a, Stx2c, and Stx2d. Regarding the late region, most of the strains carried the qO111 allele and only one strain showed differences in the ninG gene. Although the sample was limited, variability was observed in the Stx production assay, induction of the lithic cycle, Stx2 subtypes and late region of the phages, which could indicate the diversity of the phages carrying STEC strains in Colombia.