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The emergence of drug-resistant pathogens necessitates the development of new countermeasures. In this regard, the introduction of probiotics to directly attack or competitively exclude pathogens presents a useful strategy. Application of this approach requires an understanding of how a probiotic and its target pathogen interact. A key means of probiotic-pathogen interaction involves the production of small molecules called natural products (NPs). Here, we report the use of whole-cell matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry to characterize NP production by candidate probiotics (mouse airway microbiome isolates) when co-cultured with the respiratory pathogen Burkholderia. We found that a Bacillus velezensis strain inhibits growth of and elicits NP production by Burkholderia thailandensis. Dereplication of known NPs detected in the metabolome of this B. velezensis strain suggests that a previously unannotated bioactive compound is involved. Thus, we present the use of whole-cell MALDI as a broadly applicable method for screening the NP composition of microbial co-cultures; this can be combined with other -omics methods to characterize probiotic-pathogen and other microbe-microbe interactions., (© 2023 Wiley-VCH GmbH.)