Enterotoxigenic E. coli is one of the bacterial pathogens contributing to the global resistance crisis in public health and animal husbandry. The problem of antibiotic resistance is becoming more and more serious, and phage is considered one of the potential alternatives to antibiotics that could be utilized to treat bacterial infections. Our study isolated and identified a lytic phage PGX1 against multidrug-resistant enterotoxigenic E. coli EC6 strain from sewage. The phage lysis profile revealed that PGX1 exhibited a lytic effect on multidrug-resistant enterotoxigenic E. coli strains of serotype O60. Through phage whole genome sequencing and bioinformatics analysis, PGX1 was found to be the class Caudoviricetes, family Autographiviridae, genus Teseptimavirus. The length of the PGX1 genome is about 37,009 bp, containing 54 open reading frames (ORFs). Notably, phage PGX1 lacks any lysogenic-related genes or virulence genes. Furthermore, phage PGX1 demonstrates strong adaptability, tolerance, and stability in various pH (pH4-10) and temperatures (4–40°C). The in vivo and in vitro tests demonstrated that phage PGX1 significantly removes and inhibits the formation of multidrug-resistant EC6 biofilm and effectively controls the Galleria mellonella larvae and enterotoxigenic E. coli EC6 during mice infection. In conclusion, the above findings demonstrated that phage PGX1 may be a novel antimicrobial agent to control multidrug-resistant E. coli infections.