Escherichia coli – one of the most characterized bacteria and a major public health concern – remains invisible across the temporal landscape. Here, we present the meticulous reconstruction of the first ancient E. coli genome from a 16th century gallstone from an Italian mummy with chronic cholecystitis. We isolated ancient DNA and reconstructed the ancient E. coli genome. It consisted of one chromosome of 4446 genes and two putative plasmids with 52 genes. The E. coli strain belonged to the phylogroup A and an exceptionally rare sequence type 4995. The Type VI secretion system component genes appears to be horizontally acquired from Klebsiella aerogenes, however we could not identify any pathovar specific genes were detected nor any antibiotic resistances. A sepsis mouse assay performed using a closely related contemporary E. coli strain was avirulent. Our reconstruction of this ancient E. coli helps paint a more complete picture of the burden of opportunistic infections of the past.