Many studies on phage biology are based on isolation methods that may inadvertently select for narrow-host-range phages. Consequently, broad-host-range phages, whose ecological significance is largely unexplored, are consistently overlooked. To enhance research on such polyvalent phages, we developed two sequential multihost isolation methods and tested both culture-dependent and culture-independent phage libraries for broad infectivity. Lytic phages isolated from activated sludge were capable of interspecies or even interorder infectivity without a significant reduction in the efficiency of plating (0.45 to 1.15). Two polyvalent phages (PX1 of thePodoviridaefamily and PEf1 of theSiphoviridaefamily) were characterized in terms of adsorption rate (3.54 × 10⁻¹⁰to 8.53 × 10⁻¹⁰ml/min), latent time (40 to 55 min), and burst size (45 to 99 PFU/cell), using different hosts. These phages were enriched with a nonpathogenic host (Pseudomonas putidaF1 orEscherichia coliK-12) and subsequently used to infect model problematic bacteria. By using a multiplicity of infection of 10 in bacterial challenge tests, >60% lethality was observed forPseudomonas aeruginosarelative to uninfected controls. The corresponding lethality forPseudomonas syringaewas ∼50%. Overall, this work suggests that polyvalent phages may be readily isolated from the environment by using different sequential hosts, and this approach should facilitate the study of their ecological significance as well as enable novel applications.