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E. coli is one of the etiological agents responsible for pyometra in female dogs, with conventional treatment involving ovariohysterectomy. Here, we report the isolation and full characterization of two novel lytic phages, viz. vB_EcoM_Uniso11 (ph0011) and vB_EcoM_Uniso21 (ph0021). Both phages belong to the order Caudovirales and present myovirus-like morphotypes, with phage ph0011 being classified as Myoviridae genus Asteriusvirus and phage ph0021 being classified as Myoviridae genus Tequatrovirus, based on their complete genome sequences. The 348,288 bp phage ph0011 and 165,222 bp phage ph0021 genomes do not encode toxins, integrases or antimicrobial resistance genes neither depolymerases related sequences. Both phages were shown to be effective against at least twelve E. coli clinical isolates in in vitro antibacterial activity assays. Based on their features, both phages have potential for controlling pyometra infections caused by E. coli. Phage ph0011 (reduction of 4.24 log CFU/mL) was more effective than phage ph0021 (reduction of 1.90 log CFU/mL) after 12 h of incubation at MOI 1000. As a cocktail, the two phages were highly effective in reducing the bacterial load (reduction of 5.57 log CFU/mL) at MOI 100, after 12 h of treatment. Both phages were structurally and functionally stabilized in vaginal egg formulations. [ABSTRACT FROM AUTHOR]