Bacteriophages (phages), viral predators of bacteria, are an attractive way to combat the rise of antimicrobial resistance. By infecting and killing bacteria, phages generate selection pressure for the evolution of defense systems. Successfully applying phages in the clinic will, in part, depend on understanding and predicting how bacterial defense systems determine the outcomes of a phage infection. Here, we present morphological, genomic, phylogenetic, and modification-based characterization of 12 new bacteriophage species targeting Escherichia coli, isolated from water sources in Durham, UK, during undergraduate practical classes. These phages, added to our growing "Durham Collection," were all determined to be sensitive to the GmrSD-family Type IV restriction enzyme, BrxU. As such, these phages have modified genomic DNAs. HPLC and MS analysis of the genomic DNAs identified a range of modifications present in the Tequatrovirus, Krischvirus, and Mosigvirus phages, the latter of which contained 5-arabinosyl-2'-deoxycytidine (5-ara-dC) and disaccharide arabinobiose (5-ara-ara-dC) moieties. Curiously, Krischvirus phages were shown to have modification pathways distinct from those of Tequatrovirus phages. Finally, testing the modified genomic DNAs in in vitro cleavage assays with BrxU demonstrated cleavage of all modifications tested. This further extends the broad substrate specificity previously identified for BrxU. Collectively, these data provide a larger standardized Durham Collection to be used for better prediction of phage-host interactions and infection outcomes.IMPORTANCEWidespread antibiotic use has led to rising rates of antibiotic resistance. It is estimated that deaths from antibiotic-resistant bacterial infections will outpace deaths from cancer by 2050. Alternate methods of treatment are required. Bacteriophages (phages), are viruses that specifically target bacteria and are predominantly harmless to humans. There is increased interest in using phage therapy in clinics to treat infections. Studying interactions between bacteria and phages is necessary so that we can understand and better predict the outcomes of phage therapy. This will increase the chances of clinical success. Our presented work provides detailed characterization of a set of phages isolated from the environment that infect Escherichia coli, a common pathogen and model experimental system. Standardized collections of phages are time-consuming to generate and the results from our ongoing characterization of the Durham Collection presented here represent a community resource for the ease of comparison between these and other phages strains, as well as across different experimental systems.