ABSTRACTAs one of the most common pathogens of opportunistic and hospital-acquired infections,Pseudomonas aeruginosais associated with resistance to diverse antibiotics, which represents a significant challenge to current treatment modalities. Phage therapy is considered a promising alternative to conventional antimicrobials. The characterization and isolation of new bacteriophages and the concurrent evaluation of their therapeutic potential are fundamental for phage therapy. In this study, we employed an enrichment method and a double-layer agar overlay to isolate bacteriophages that infectP. aeruginosastrains PAO1 and PA14. Three phages (named PA_LZ01, PA_LZ02, and PA_LZ03) were isolated and showed icosahedral heads and contractile tails. Following full-genome sequencing, we found that phage PA_LZ01 contained a genome of 65,367 bp in size and harbored 90 predicted open reading frames (ORFs), phage PA_LZ02 contained a genome of 57,243 bp in size and harbored 75 predicted ORFs, and phage PA_LZ03 contained a genome of 57,367 bp in size and carried 77 predicted ORFs. Further comparative analysis showed that phage PA_LZ01 belonged to the genusPbunavirusgenus, phage PA_LZ02 belonged to the genusPamexvirus, and phage PA_LZ03 belonged to the familyMesyanzhinovviridae. Next, we demonstrated that these phages were rather stable at different temperatures and pHs. One-step growth curves showed that the burst size of PA_LZ01 was 15 PFU/infected cell, and that of PA_LZ02 was 50 PFU/infected cell, while the titer of PA_LZ03 was not elevated. Similarly, the biofilm clearance capacities of PA_LZ01 and PA_LZ02 were also higher than that of PA_LZ03. Therapeutically, PA_LZ01 and PA_LZ02 treatment led to decreased bacterial loads and inflammatory responses in a mouse model. In conclusion, we isolated three phages that can infectP. aeruginosa, which were stable in different environments and could reduce bacterial biofilms, suggesting their potential as promising candidates to treatP. aeruginosainfections.IMPORTANCEPhage therapy is a promising therapeutic option for treating bacterial infections that do not respond to common antimicrobial treatments. Biofilm-mediated infections are particularly difficult to treat with traditional antibiotics, and the emergence of antibiotic-resistant strains has further complicated the situation.Pseudomonas aeruginosais a bacterial pathogen that causes chronic infections and is highly resistant to many antibiotics. The library of phages that targetP. aeruginosais expanding, and the isolation of new bacteriophages is constantly required. In this study, three bacteriophages that could infectP. aeruginosawere isolated, and their biological characteristics were investigated. In particular, the isolated phages are capable of reducing biofilms formed byP. aeruginosa. Further analysis indicates that treatment with PA_LZ01 and PA_LZ02 phages reduces bacterial loads and inflammatory responsesin vivo. This study isolated and characterized bacteriophages that could infectP. aeruginosa, which offers a resource for phage therapy.