BACKGROUND: The chewing stick (miswak) is used for oral hygiene in many parts of the world. In addition to the mechanical removal of plaque, an antibacterial effect has been postulated; however, tests of miswak extract from Salvadora persica (Arak) disclosed only low to moderate antibacterial effects. This may be attributable to the extraction process. Our aim was to test in vitro the antibacterial effect of miswak pieces, without extraction, on bacteria implicated in the etiology of periodontitis and caries. METHODS: Miswak pieces were standardized by size and weight (0.07 and 0.14 g) and tested against Streptococcus mutans, Lactobacillus acidophilus, Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), Porphyromonas gingivalis, and, as a reference, Haemophilus influenzae. The miswak pieces were tested in two ways: embedded in the agar plate or suspended above the agar plate. RESULTS: The inhibitory effect was most pronounced on P. gingivalis, A. actinomycetemcomitans, and H. influenzae, less on S. mutans, and least on L. acidophilus. Suspended miswak had comparable or stronger effects than miswak embedded in agar. The 0.14-g suspended miswak exhibited significantly greater inhibition on A. actinomycetemcomitans and H. influenzae than the 0.14-g miswak embedded in agar (P<0.01 and P<0.001, respectively). CONCLUSIONS: Miswak embedded in agar or suspended above the agar plate had strong antibacterial effects against all bacteria tested. The antibacterial effect of suspended miswak pieces suggests the presence of volatile active antibacterial compounds.