This study aimed to count Enterobacteriaceae and Escherichia coli in different locations on pig carcasses (shank, loin, abdomen, shoulder, and jowl) from two slaughterhouses (A and B) between September 2019 and July 2021 during different slaughter stages (after bleeding, after passing through the epilator machine, after manual toileting in the dirty area, before and after evisceration, and after the final washing), as well as verify antimicrobial resistance and biofilm formation capacity. The main points of Enterobacteriaceae and E. coli contamination were identified in the two slaughterhouses through three collections. The stages with the highest counts were post-bleeding and evisceration in both slaughterhouses and after manual toileting in slaughterhouse B in the first collection. Most E. coli isolates were resistant to multiple antimicrobials, with higher resistance frequencies to amoxicillin, ampicillin, chloramphenicol, sulfonamides, and streptomycin. The virulence genes eae, stx1, and stx2 were also detected. Three isolates had all three genes and exhibited resistance to at least six antimicrobial classes (β-lactams, macrolides, aminoglycosides, sulfonamides, amphenicols, and quinolones). E. coli isolates also showed a high frequency of strains with moderate and strong in vitro biofilm-forming capacity. This is the first study to characterize microbial contamination by pig slaughter stage in the Federal District region, demonstrating the critical points for hygienic production. E. coli was isolated from the surface of pig carcasses, as well as the virulence genes stx1, stx2, and eae were detected. The multi-antimicrobial resistant isolates also had a moderate-to-strong biofilm formation capacity, thus demonstrating risks to public health.