Isolate 18191, obtained from mature strawberry fruit and determined as Paenibacillus polymyxa has shown an antagonistic potential against Botrytis cinerea, the causal agent of grey mould in strawberries. Germ tube growth of conidia of B. cinerea was strongly inhibited by the culture suspension of the antagonist in aqueous strawberry fruit pulp suspension (1%) but germination rate of conidia was not affected. The application of the culture suspension and the washed cells on detached strawberry leaf discs reduced conidiophore density of B. cinerea by 67 and 84%, respectively. The treatment of detached leaf discs with culture suspensions of different cell densities (1 × 106, 1 × 107, 1 × 108) showed that the lowest density already reduced incidence of B. cinerea by 68% after 8 days incubation period. Investigating the influence of the temperature on the effectiveness of P. polymyxa it was observed that the antagonist was highly effective already at 10°C and reduced incidence and conidiophore density of B. cinerea by 53 and 58%, respectively. In 3-year field trials the effectiveness of P. polymyxa was in a range of 24–36% as compared to the water control.