Background. Hand hygiene is a fundamental component of infection control. Hand contamination with Staphylococcus aureus andEscherichia coli may contribute to infections. Objectives. To assess the effectiveness of different handwashing methods in reducing the levels of bacterial flora, especially S. aureus and E. coli, on the hands of theatre staff. Methods. A cross-sectional study was conducted among 70 staff in surgical theatres of three randomly chosen hospitals in Johannesburg, South Africa. Samples were taken before and after handwashing using the modified glove juice method and the fingernail press technique. Standard microbiological techniques were used to identify bacteria. Descriptive statistics and non-parametric analysis were used to compare the differences between hospitals and to determine the effects of handwashing on microbial flora and skin irritation. Results. S. aureus organisms were isolated in the prewash samples of 29 (41%) and in the postwash samples of 20 (29%) workers. Of the 29 with positive prewash cultures, 19 (65.5%) showed decreased postwash counts, while 10 (34.5%) showed no change or increased counts. Four workers with a negative prewash count had a positive postwash count. No statistical differences were found between postwash counts categorised by the type of cleansing formula used and the washing technique. E. coli organisms were identified in the prewash count of the fingertip press of one worker. Conclusions. Almost half of the theatre staff carried S. aureus isolates on their hands prior to handwashing and approximately one-third after handwashing. Closer monitoring of handwashing techniques should be introduced.