Escherichia coli sequence type 131 (E. coli ST131) is an important pathogen of nosocomial and communal urinary tract infections and bacteremia. Rapid dissemination of multidrug-resistant (MDR) E. coli ST131 clade C is becoming a global burden with limited alternative treatment options. Genetically diverse bacteriophages (phages), the virus that kills bacteria, isolated from wastewater have demonstrated lytic activity against various pathogens, including E. coli ST131. Additionally, phage treatment has shown a promising outcome in patients infected with MDR pathogens over recent years. In our study, we isolated lytic phages (n = 22) from wastewater (n = 20) collected from four hospitals in Calgary, Alberta. We also assessed intergenomic similarity/difference of isolated lytic phages using VIRIDIC (Virus Intergenomic Distance Calculator). Of the 22 phages, 11 genetically different phages were used for host range screening to determine their lytic activity against clade C, consisting of subclades C1 (n = 8), associated with fluoroquinolone-resistance, C1-M27 (n =9), harbouring extended-spectrum β-lactamase (ESBL), and C2 (n = 8), associated with ESBL CTX-M-14. The analysis showed that 16% (n = 3) to 60% (n = 15) of clade C strains were susceptible to each phage, with two subclade C2 strains resistant to all phages. Furthermore, our preliminary data on genome-wide association studies (GWAS) via Scoary revealed that surface proteins, such as outer membrane usher protein, lipoprotein, pilin, and fimbrial-associated proteins may be pertinent to phage susceptibility. Overall, genetically different lytic phages isolated from hospital wastewater have a broad host range against E. coli ST131 clade C clones, and various host surface proteins may serve as receptor for these phages.