The carbapenem-resistant Enterobacterales (CRE) poses a pressing public health concern. Here, we investigated the frequency of CRE bacteria, carbapenemase-encoding genes, and the molecular epidemiology of carbapenemase-resistant Escherichia coli in wastewater resources and healthy carriers in Iran. Out of 617 Enterobacterales bacteria, 24% were carbapenem-resistant. The prevalence of CRE bacteria in livestock and poultry wastewater at 34% and hospital wastewater at 33% were significantly higher (P ≤ 0.05) compared to those in healthy carriers and municipal wastewater at 22 and 17%, respectively. The overall colonization rate of CRE in healthy individuals was 22%. Regarding individual Enterobacterales species, the following percentages of isolates were found to be CRE: E. coli (18%), Citrobacter spp. (24%), K. pneumoniae (28%), Proteus spp. (40%), Enterobacter spp. (25%), Yersinia spp. (17%), Hafnia spp. (31%), Providencia spp. (21%), and Serratia spp. (36%). The blaOXA-48 gene was detected in 97% of CRE isolates, while the blaNDM and blaVIM genes were detected in 24 and 3% of isolates, respectively. The B2 phylogroup was the most prominent group identified in carbapenem-resistant E. coli isolates accounting for 80% of isolates. The presence of CRE bacteria carrying transmissible carbapenemase-encoding genes among healthy individuals and wastewater resources in Iran underscores the more assertive measures to prevent the CRE spread.