Bacteriophages (phages) are ubiquitous viruses which have adapted to infect and replicate within target bacteria, their only known hosts, in a strain specific fashion with minimal cross infectivity. The recent steep rise in antibiotic resistance throughout the world has renewed interest in adapting phages for the imaging and treatment of bacterial infection in humans. In this article, we describe the current limitations surrounding the radiolabeling of phage for the imaging and treatment of bacterial infection and methods to overcome these difficulties. Specifically, we examined the effects of hydrazinonicotinamide conjugation and removal of bacterial DNA on the infectivity, biodistribution, and radionuclide imaging of a phage lytic for a clinically relevant strain of Pseudomonas aeruginosa, a common Gram-negative bacterial pathogen often resistant to multiple antibiotics. We found that all but the briefest reaction of concentrated phage with hydrazinonicotinamide (≤3 min) resulted in nearly complete loss of infectivity. Furthermore, we determined that digestion and removal of bacterial DNA was needed to avoid high nonspecific uptake of hydrazinonicotinamide-labeled phage within the liver and spleen as well as prolonged circulation in the blood. We also demonstrate the surprisingly wide soft tissue and organ biodistribution and rapid pharmacokinetics of 99mTc-hydrazinonicotinamide-labeled phage in normal mice as well as its imaging characteristics and efficacy in wounded mice infected with bioluminescent Pseudomonas aeruginosa. In conclusion, the preservation of phage infectivity and removal of all bacterial containments including DNA are critical methodologic considerations in the labeling of phages for imaging and therapy.