The use of supernatant from Bacillus spp. as biological control agents has potential to manage strawberry fruit rot caused by Botrytis cinerea. This study aimed to identify the most effective Bacillus spp. supernatant in suppressing the growth of B. cinerea both in vitro and in vivo. The research consisted of two stages: (1) evaluation of the inhibitory effect of Bacillus spp. supernatant on the in vitro growth of B. cinerea, and (2) assessment of their ability to suppress strawberry fruit rot development in vivo. A Completely Randomized Design (CRD) was applied with six treatments and five replications for the in vitro test, and five treatments with five replications for the in vivo test. The results showed that supernatant of Bacillus cereus Se07, Bacillus cereus P14, and Bacillus subtilis effectively inhibited the in vitro growth of B. cinerea, producing colony areas of 33.13 cm², 30.18 cm², and 27.74 cm², respectively, with inhibition rates of 46.30%, 51.08%, and 55.04%, categorized as strong inhibition. Reduced colony growth was associated with decreased conidial germination, with the lowest germination rate (17.31%) observed in the Bacillus subtilis treatment. In vivo, Bacillus subtilis also produced the smallest lesion area on strawberry fruit (6.02 mm²).
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