Meticillin-resistant Staphylococcus aureus (MRSA) is a major pathogen responsible for significant numbers of healthcare-associated infections and isolates containing Panton-Valentine leukocidin (PVL) that cause severe skin infections are emerging as a serious problem. The rapid detection of MRSA would be an invaluable tool in a diagnostic laboratory. The aim of this study is to develop real-time polymerase chain reaction (PCR) assays for the detection of MRSA and PVL directly from clinical samples, and then combining these assays. Individual assays for MRSA (SCCmec) and PVL (lukF and lukS) were optimised and evaluated with screening and wound swabs, respectively. MRSA- and PVL-positive isolates were detected by the assays with an analytical sensitivity of 100 cfu per reaction. No other bacterial species were amplified. Fifty of 402 (12.4%) nasal swabs were positive by culture and PCR. Four of the 402 (1.0%) swabs were PCR-positive/culture-negative. Three of the 402 (0.7%) swabs were PCR-negative/culture-positive. The sensitivity of the MRSA assay is 95% and the specificity is 99% using conventional culture as the gold standard. Five of 240 wound swabs (2.1%) were positive for PVL. Three of the PVL-positive swabs were meticillin-sensitive Staphylococcus aureus (MSSA) and two were MRSA. The MRSA assay is a powerful and sensitive diagnostic tool, giving rapid results and could allow more timely treatment and infection control decisions to be taken. It can also, when combined with the PVL assay, provide valuable epidemiological information.