Shiga toxin-producing Escherichia coli (STEC) O157:H7 and extended-spectrum beta-lactamase (ESBL) producing E. coli (ESBLEC) are important bacteria of public health concern and frequently isolated from raw beef products. Bacteriophage-based methods have been increasingly exploited to control bacterial contamination in meats. Here, we describe the isolation, characterization, and application of a lytic phage PE37 for the simultaneous bio-control of STEC O157:H7 and ESBLEC. Phage PE37, isolated from the bovine intestine, was morphologically characterized as a member of the Myoviridae family, with a broad host range and great stability under various stress conditions. Sequencing analysis revealed that the genomic DNA of phage PE37 contains genes that contribute to virion structure, replication, assembly, and host lysis. PE37 significantly reduced the viable counts of STEC O157:H7 by 4.9 and 2.6logCFU/mL in broth after 6h of incubation at 25 and 8°C, respectively. Application of phage PE37 to raw beef artificially contaminated with STEC O157:H7 resulted in significant reductions in the viable counts by 2.3 and 0.9logCFU/piece after 24h of storage at 25 and 8°C, respectively. Treatment of raw beef contaminated with a bacterial cocktail of STEC O157:H7 and ESBLEC with PE37 also significantly decreased the viable counts of the bacterial mixture by 1.4 and 1.0logCFU/piece after 24h of incubation at 25 and 8°C, respectively. These findings suggest that bacteriophage PE37 may be a potential bio-agent for controlling STEC O157:H7 and ESBLEC contamination in raw beef.