The aim of the study was to determine the prevalence of Pseudomonas aeruginosa and Klebsiella pneumoniae strains in patients with acute leukemias, to assess their clinical significance, and to define the sources and ways of their spread using genetic analysis. Thirty-four patients were investigated during the observed period. Twenty-one strains of Pseudomonas aeruginosa and 35 strains of Klebsiella pneumoniae were isolated from patient samples. In the case of Pseudomonas aeruginosa, 47.6% of strains were identified as pathogens and caused infection. By contrast, only 4 isolates (11.4%) of Klebsiella pneumoniae could be regarded as etiological agents of bacterial infection. Based on the obtained results, Klebsiella pneumoniae strains are assumed to be of mostly endogenous origin. In the case of Pseudomonas aeruginosa strains, the proportion of identical strains detected in various patients was higher and exogenous sources were more significant. In addition, our results confirmed the ability of Pseudomonas aeruginosa strains to survive on a particular site in the hospital for a longer time.