This study assessed the prevalence and zoonotic potential of Shiga toxin-producing Escherichia coli (STEC) sampled from 104 dairy units in the central region of Zambia and compared these with isolates from patients presenting with diarrhoea in the same region. A subset of 297 E. coli strains were sequenced allowing in silico analyses of phylo-and sero-groups. The majority of the bovine strains clustered in the B1 'commensal' phylogroup (67%) and included a diverse array of serogroups. 11% (41/371) of the isolates from Zambian dairy cattle contained Shiga toxin genes (stx) while none (0/73) of the human isolates were positive. While the toxicity of a subset of these isolates was demonstrated, none of the randomly selected STEC belonged to key serogroups associated with human disease and none encoded a type 3 secretion system synonymous with typical enterohaemorrhagic strains. Positive selection for E. coli O157:H7 across the farms identified only one positive isolate again indicating this serotype is rare in these animals. In summary, while Stx-encoding E. coli strains are common in this dairy population, the majority of these strains are unlikely to cause disease in humans. However, the threat remains of the emergence of strains virulent to humans from this reservoir.