Colibacillosis in chickens can cause the death of young stock, decrease weight gain and lead to significant economic losses. Currently, antibiotic therapy is the main method of treatment of infected animals, but unchecked use of antibiotics has led to widespread antibiotic resistance among microorganisms. Therefore, it is necessary to develop alternative methods of treating bacterial infections that are fully consistent with the One Health concept and introduce them into practice. Phage therapy meets the specified requirements perfectly. This study describes the isolation and characterization of the lytic jumbo phage vB_EcoM_Lh1B and evaluates its potential use in controlling antibiotic-resistantE. coliinfection in poultry. The complete phage genome is 240,200 bp long. Open reading frame (ORF) prediction shows that the phage genome does not contain genes encoding antibiotic resistance and lysogeny factors. Based on phylogenetic and electron microscopic analysis, vB_EcoM_Lh1B belongs to the group of myoviruses of theSeoulvirusgenus of theCaudoviricetesclass. The bacteriophage has good resistance to a wide range of pH and temperatures and has the ability to suppress 19 out of 30 studied pathogenicE. colistrains. The biological and lytic properties of the isolated vB_EcoM_Lh1B phage make it a promising target of further study as a therapeutic agent againstE. coliinfections in poultry.