The characterization of Shiga toxin-producing Escherichia coli (STEC) is necessary to assess their pathogenic potential, but isolation of the strain from complex matrices such as milk remains challenging. In previous work, we have shown the potential of long-read metagenomics to characterize eae-positive STEC from artificially contaminated raw milk without isolating the strain. The presence of multiple E. coli strains in the sample was shown to potentially hinder the correct characterization of the STEC strain. Here, we aimed at determining the STEC:commensal ratio that would prevent the characterization of the STEC. We artificially contaminated pasteurized milk with different ratios of an eae-positive STEC and a commensal E. coli and applied the method previously developed. Results showed that the STEC strain growth was better than the commensal E. coli after enrichment in acriflavine-supplemented BPW. The STEC was successfully characterized in all samples with at least 10 times more STEC post-enrichment compared to the commensal E. coli. However, the presence of equivalent proportions of STEC and commensal E. coli prevented the full characterization of the STEC strain. This study confirms the potential of long-read metagenomics for STEC characterization in an isolation-free manner while refining its limit regarding the presence of background E. coli strains.