Burkholderia gladioli has only recently been reported to be a human pathogen. Four cases of B. gladioli infection (including bacteremia, pneumonia, and cervical adenitis) in two adults and two young children are reported. Three of these four patients were severely immunocompromised. Commercial systems were frequently unable to identify this bacterium correctly. Antimicrobial susceptibility patterns indicated that B. gladioli strains were susceptible to the quinolones, aminoglycosides, and imipenem. In vitro laboratory investigations demonstrated that B. gladioli strains were susceptible to complement-mediated lysis of pooled human serum, thus implying that healthy individuals should be immune to infection. These four cases together with three previously reported cases suggest that B. gladioli primarily causes disease in severely immunocompromised individuals. The lack of mortality associated with infection, coupled with susceptibility to serum and lack of recognizable virulence-associated factors, suggests that this species has a low pathogenic potential.