Pseudomonas aeruginosa is an opportunistic pathogen which is considered as one of the important causes of hospital-acquired infections such as pneumonia, wound infections, urogenital and intra-abdominal sepsis. Phages are hopeful alternatives for antibiotics and they can eliminate the biofilms and pathogens effectively. The objectives of this work are assessing the characterization and anti-biofilm activity of a naturally isolated phage on P. aeruginosa. Morphological analysis by transmission electron microscopy revealed the isolated phage is an icosahedral particle with ~50 nm diameter The isolated phage has a species-specific host range for P. aeruginosa. The analysis of the phage proteins resulted in a pattern with about 10 proteins and a major band of ~35 kDa. The phage exhibited 86.82 and 86.86% biofilm inhibition at PFU of 10 and 100, respectively. Furthermore, the phage is effective on some antibiotic resistant clinically-isolated P. aeruginosa. The results of this study shows that the natural phages are effective agents against biofilms. Due to increasing resistant pathogens to antibiotics, alternative agents such as phages are promising tools for treating bacterial infections.