In laying and breeding chickens, pathomorphological signs of histomonosis often coincide with colibacillosis. Thus, we investigated the systemic spread of Escherichia coli in chickens affected with histomonosis and colibacillosis by characterizing their pheno- and genotypic profiles. For this, 29 birds from 11 affected flocks were necropsied and up to three E. coli isolates each from intestine, heart, and liver of the birds were isolated. A total of 251 isolates were characterized by serotyping, phylogenetic grouping, detection of virulence-associated genes (VAGs), and pulsed-field gel electrophoresis (PFGE). All birds showed egg peritonitis, and fibrinous typhlitis was additionally recorded in 18 birds. Presence of Histomonas meleagridis in ceca was confirmed by PCR and histopathology. Escherichia coli serotype O2:K1 was found to be the most prevalent (37.4%), whereas 31.1% of strains were not typeable. The majority of isolates collected from the intestine and extraintestinal organs belonged to phylogroups B2 (54.1%), D (21.5%), or A (19.5%). Isolates from these phylogroups harbored a higher number of VAGs. Macrorestriction analysis showed that 60.6% of total isolates from all organs tested were included in eight PFGE types. Isolation of E. coli with identical genomic profiles from the intestine and extraintestinal organs of the same or different birds in the same flock indicates for systemic dissemination of the bacteria, independent of E. coli genotype. Intestinal destruction due to H. meleagridis can be considered as the most plausible cause of bacterial dissemination, ultimately leading to colibacillosis.