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• Two new virulent phage vB_EcoS_XF and vB_EcoS_XY2 were isolated, characterized and compared. • Whole genome analysis indicated that no known virulence-related or antibiotic resistance genes exist. • No obvious cytotoxicity was observed when either HEK293 T or A549 cells were incubated with vB_EcoS_XF and vB_EcoS_XY2. • vB_EcoS_XF and vB_EcoS_XY2 are new virulent phages belonging to the Kagunavirus genus, which can infect their host specifically. Escherichia coli is an opportunistic bacterial pathogen that causes a wide range of nosocomial infections. The emergence of multidrug resistance in E. coli poses a severe threat to global health. Phage therapies are an alternative method to control multidrug-resistant pathogens, which have been attracting increasing attention. Owing to their ability to lyse bacteria specifically and efficiently, bacteriophages are considered novel antimicrobial agents. In this study, we used multidrug-resistant E. coli as an indicator and isolated, characterized, and compared two new phages of the Siphoviridae family referred to as vB_EcoS_XF and vB_EcoS_XY2. These phages were able to infect several pathogenic multidrug-resistant E. coli strains. A short latent period and large burst size ensured their rapidly reproduction in host cells. Their tolerance of high temperatures and high pH levels meant that remained stable when used to control pathogenic E. coli strains. No obvious cytotoxicity was observed when either HEK293 T or A549 cells were incubated with these two phages. Mass spectrometry analysis allowed us to identify several phage-encoded proteins. Genomic analysis revealed that no toxic proteins or antibiotic proteins were encoded. Genome comparison and phylogenetic analysis indicated that the phages identified show high similarity with E. coli phages of the genus Kagunavirus. The desirable characteristics of the novel phages identified make them good potential therapeutic candidates, and components of phage cocktails to treat multidrug-resistant E. coli in the future. [ABSTRACT FROM AUTHOR]