Tea tree oil has recently emerged as an effective topical antimicrobial agent active against a wide range of organisms. Tea tree oil may have a clinical application in both the hospital and community, especially for clearance of methicillin-resistant Staphylococcus aureus (MRSA) carriage or as a hand disinfectant to prevent cross-infection with Gram-positive and Gramnegative epidemic organisms. Our study, based on the time-kill approach, determined the kill rate of tea tree oil against several multidrug-resistant organisms, including MRSA, glycopeptide-resistant enterococci, aminoglycoside-resistant klebsiellae, Pseudomonas aeruginosa and Stenotrophomonas maltophilia, and also against sensitive microorganisms. The study was performed with two chemically different tea tree oils. One was a standard oil and the other was Clone 88 extracted from a specially bred tree, which has been selected and bred for increased activity and decreased skin irritation. Our results confirm that the cloned oil had increased antimicrobial activity when compared with the standard oil. Most results indicated that the susceptibility pattern and Gram reaction of the organism did not influence the kill rate. A rapid killing time (less than 60 min) was achieved with both tea tree oils with most isolates, but MRSA was killed more slowly than other organisms.