Aims: To assess the activity of Brevibacillus brevis (formerly Bacillus brevis) Nagano and the antibiotic it produces, gramicidin S, against the plant pathogen Botrytis cinerea. Methods and Results: Germination and growth of Bot. cinerea were assessed in the presence of B. brevis or gramicidin S in liquid media, on solid media and on leaf sections of Chinese cabbage. Germination was 10-fold more sensitive to gramicidin S than growth. Inhibition of Bot. cinerea was greater in liquid media compared with on solid media. Activity of gramicidin S against Bot. cinerea on leaf sections was much lower than in vitro. In vitro inhibition of Bot. cinerea by B. brevis Nagano was similar to equivalent levels of gramicidin S. Conclusions: Antibiosis, via gramicidin S, is the mode of antagonism exhibited by B. brevis Nagano against Bot. cinerea in vitro. Significance and Impact of the Study: The mode of antagonism of B. brevis against Bot. cinerea was elucidated. The differing activity of gramicidin S against Bot. cinerea in vitro and on leaf sections indicates one mechanism by which biocontrol activity may differ between laboratory and field conditions.