Escherichia Coli ( E. coli) is one of the most prevalent bacteria in the gut and plays an important role in the formation of the intestinal microbiome. In this study, Escherichia phage Ioannina, a novel member of Caudoviricetes, with high lytic activity against E. coli ATCC 25922, was isolated from hospital wastewater. Escherichia phage Ioannina was highly resistant to a broad range of temperatures and alkaline pH, but was sensitive to acidic pH. Whole genome sequencing of the phage, revealed that Escherichia phage Ioannina is a novel phage within the Dhillonvirus genus. Its genome is a 45,270 bp double-stranded DNA molecule that encodes 64 putative open reading frames (ORFs). The phylogenetic analysis of open reading frames, which diversified Escherichia phage Ioannina from other dhillonviruses, indicated that ORF31 (a putative tail fiber protein) presented higher similarity to representatives of other phage families. ORF31 was significantly more related to similar ORFs belonging to the Kuravirus and Tunavirus ( Drexlerviridae) genera, indicating a possible recombination event.