Bioactive molecules produced by microorganisms during submerged fermentation could be of interest to develop new biopesticides. In this work, we identified and evaluated the biological activity of metabolites produced by Metarhizium robertsii Mt004 during microsclerotia formation in submerged fermentation. For this, the cell-free supernatant obtained after fermentation was subjected to liquid–liquid extraction using a 1:1 mixture of dichloromethane and ethyl acetate. The resulting organic (OF) and aqueous fractions (WF) were subsequently tested for insecticidal activity through bioassays with Diatraea saccharalis larvae. Fractions (OF and cell free supernatant) demonstrating activity were selected for chemical analysis via high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Both the cell-free supernatant and the organic fraction caused larvae mortality (48%). The data obtained from the mass spectrometry analysis of the organic fraction were used to build molecular networks using the Global Natural Products Social Molecular Networking (GNPS) platform. Most compounds in the organic fraction were from the cyclodepsipeptide family, suggesting the presence of 17 destruxins, including destruxin A, A2 and B, all recognised for their insecticidal activity. Therefore, the microsclerotia and the metabolites produced during the fermentation caused insect mortality, suggesting that both resources can be exploited to develop new microbial products for pest control.