In healthy volunteers, surgical hand rubbing with Sterillium^® for 1.5min has been shown to be as effective as a 3min procedure. The aim of this study was to assess whether this result was reproducible under in-use conditions. During nine weeks in the ambulatory surgery theatre of a 750-bed tertiary care university hospital, the two surgical hand-rubbing procedures were compared with each other, and with a hand-scrubbing procedure using a povidone-iodine (4%) scrub prior to and after 25 different surgical operations for each. Imprints of the surgeon’s dominant hand were taken on culture plates before and within 1min following the end of the hand-rubbing/scrubbing procedures (immediate effect) and at the end of surgery (sustained effect). Plates were incubated aerobically at 37°C for 48h. Colonies were counted at 24h and 48h. Results were expressed as the number of colony-forming units per hand. No significant difference in baseline hand bacterial load was found before the hand-rubbing/scrubbing procedures among the three groups (P=0.19). With respect to immediate and sustained antimicrobial effects, a significantly greater reduction in microbial loads on the hands was achieved with the 3min hand-rubbing protocol as opposed to hand-scrubbing protocol (P=0.04 and P=0.02, respectively), but there was no difference between the reductions obtained with 1.5 and 3min rubbing protocols (P=0.41 and P=0.36, respectively). Surgical hand rubbing with Sterillium using a 1.5min protocol should be considered as an attractive alternative method for surgical hand disinfection.