The aim of this study was to investigate whether the in vitro presence of blood cells influences the anti-microbial activity of hyperbaric oxygen (HBO) against Escherichia coli. Fifty E. coli isolates from clinical samples were used in the study. A small number of colonies belonging to each isolate from the nutrient media were transferred into two K(3)EDTA tubes (the blood group) and two Mueller-Hinton broth tubes (the broth group). Then, both groups were divided into subgroups according to whether HBO was administered (HBO subgroup) or not (non-HBO subgroup). HBO treatment was applied for one hour at 2.5 absolute atmospheres. The tubes in the non-HBO subgroup were left at room temperature during this period. Subsequently, all the tubes were cultured on Mueller-Hinton and Eosin Methylene Blue agar using the quantitative counting technique. After 18 to 24 h incubation at 37 degrees C, the colonies formed in the plates were counted. In the blood group, compared with non-HBO subgroup samples, the number of colonies decreased in 56% of samples, increased in 32% of samples and did not change in 12% of samples in the HBO subgroup. Whereas, in the broth group the number of colonies decreased in only 32% of samples increased in 38% of samples and did not change in 30% of samples in the HBO subgroup compared with the non-HBO subgroup. The difference between the blood and the broth groups revealed a statistical significance using Pearson's Chi-square test (P = 0.025). We concluded that the antibacterial effect of HBO on E. coli increases in the cellular environment belonging to the host organism.