Abstract Larval frass from insects which consists of larval excrement, exoskeleton, and undigested diet, is a rich source of organic material and microorganisms. Despite its potential value, research on frass valorisation in agriculture is limited. In this study, single-layer agar (SLA) and double-layer agar (DLA) in vitro bioassays were conducted to evaluate the effect of water-based black soldier fly larvae (BSFL) frass extracts from two different diets on the growth of six plant pathogens: Alternaria solani, Botrytis cinerea, Fusarium oxysporum, Phytophthora capsici, Rhizoctonia solani and Sclerotinia sclerotiorum. The results showed that frass extract from Gainesville house fly diet strongly or completely inhibited the growth of all tested plant pathogens in both SLA and DLA bioassays, while frass extract from fruit/vegetable/bakery/brewery diet strongly inhibited the mycelial growth of A. solani, B. cinerea, and S. sclerotiorum, and moderately inhibited the mycelial growth of P. capsici in both bioassays. For both diets and bioassays, 0.22 μm microfiltered frass extracts which are free of microorganisms showed generally no effect on the growth of the pathogens indicating that growth inhibition is caused by frass-inhabiting microorganisms. Both SLA and DLA bioassays revealed strong antagonistic effect of microorganisms inhabiting BSFL frass against the plant pathogens B. cinerea, A. solani, R. solani, P. capsici, F. oxysporum and S. sclerotiorum. Moreover, the study showed the impact of the BSFL diet on the antagonistic effect of frass extract. In future work, the antagonistic effect of frass extracts against the above-mentioned pathogens will be tested in vivo. BSFL frass could eventually find applications for the control of plant diseases.