This study investigated in-vitro, ex-vivo and in-vivo the immunomodulatory effects of rufloxacin. 0.5 MIC of rufloxacin significantly enhanced human macrophage phagocytosis and increased intracellular killing of Klebsiella pneumoniae in vitro. Pre-incubation of K. pneumoniae with rufloxacin made the bacteria more susceptible to both phagocytosis and intracellular killing by human macrophages than control organisms. Following pre-exposure of macrophages to 0.5 MIC of rufloxacin, there was a significant increase in the intracellular killing of K. pneumoniae compared with the controls, indicating the ability of rufloxacin to cross biological membranes and to remain active within phagocytes. Ex-vivo experiments show that iv administration of rufloxacin in mice lead to an increase in both phagocytic and microbicidal intracellular activity by phagocytes. In-vivo models of experimental infections showed that prophylactic administration of rufloxacin increased the survival of mice after challenge with Candida albicans.