The use of bacteriophages, viruses that infect bacteria, in the treatment of multidrug resistant (MDR) infections is being increasingly regarded as an alternative to antibiotics. However, many questions regarding best clinical protocols are still debated. A promising clinical avenue is personalized therapy based on ‘magistral’ bacteriophage preparations that rely on the availability of large, well-characterized phage banks accessible ad hoc for patient care. For this purpose continued isolation and characterization of lytic phages against clinically relevant pathogens is critical. We have used modified standard methods for phage isolation to build an exhaustive phage library against MDR pathogens, in particular, but not exclusively, those related to severe infections (E. coli, K. pneumoniae, S. aureus, Enterococcus ssp., Pseudomonas aeruginosa). To maximize isolation frequencies, our protocols included use of both enrichment and direct plating, varying agar concentrations for growth of phages that do not perform well under regular conditions, extended enrichment incubations, as well as multiple environmental sources for isolation (soil, water, wastewater, hair, skin, sputum, faeces). We isolated phages against common human pathogens (“high risk clones” E. coli ST131 and K. pneumoniae ST258, MDR P. aeruginosa and methicillin resistant and sensitive S. aureus) but with different rates of success and host specificities. For K. pneumoniae and E. coli we isolated diverse lytic phages, while for other bacterial species such as S. aureus standard isolation methods had poor success with frequent selection of temperate phages (not useful for immediate therapeutic applications). Differential results in phage isolation with standard methodology are dependent on the bacterial host genomic background, related to phage resistance mechanisms and species genomic variability, as well as to phage biology and bacteria-phage population dynamics. Bacteriophage isolation is usually not considered as a primary bottleneck for therapy, but this holds true only for a selected number of bacterial targets. There is a need for further study in order to improve isolation techniques to effectively select for virulent phages of clinical interest.