Summary Globally, root-knot nematode (RKN) infestations cause great financial losses. Although agrochemicals are used to manage these pests, there is increased interest in using biocontrol agents based on natural antagonistic microorganisms, such as Bacillus. These nematicidal bacteria demonstrate antagonism towards RKN through different modes of action, including specialised metabolite production. The aim of this study was to compare metabolite profiles of nematicidal Bacillus species and assess the influence of cultivation conditions on these profiles. Two hyphenated metabolomics platforms, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF-MS), were employed to profile and compare metabolite features produced during the cultivation of three nematicidal Bacillus species (Bacillus firmus, B. cereus and B. soli) in complex Luria-Bertani broth (LB) and a simpler minimal broth (MB), at three different temperatures (25, 30 and 37°C). Cultivation in complex LB as opposed to simpler MB resulted in the production of more statistically significant metabolite features. Selected temperatures in this study did not have a significant influence on metabolite profiles. Moreover, media-specific influences outweighed temperature-specific influences on metabolite profiles. Results from this study are a valuable first step in establishing suitable cultivation conditions for the production of Bacillus metabolites of interest.