Fungal co-culture has several biotechnological applications including the discovery or the enhanced production of secondary metabolites. It is also a powerful tool aiding to elucidate the involvement of secondary metabolism in fungus-fungus interactions. The aim of this work was to investigate secondary metabolites produced when Fusarium verticillioides is co-cultured with Gliocladium roseum. Secreted metabolites were analyzed by HPLC-MS, and fusaric acid (FA) was quantified by HPLC-DAD. Four FA derivatives were identified only in the F. verticillioides-G. roseum co-culture. Mass spectrometry and one- and two-dimensional NMR spectra indicated that they were 5-butylpyridine-2-carboxylic acid methyl ester (5B2CAM), 4-(5-butylpicolinamido) butanoic acid (45BBA), methyl 4-(5-butylpicolinamido) butanoate (M45BBA), and bis(5-butyl-2-pyridinecarboxylate-N1,O2)-copper (B52P). 45BBA and M45BBA are reported for the first time and were FA biotransformation products generated by G. roseum. The antifungal activity of 5B2CAM, 45BBA, and M45BBA was evaluated in vitro against Botrytis cinerea and Aspergillus niger. They were less fungitoxic than FA, with 45BBA as the least toxic. Our results suggest that the effective antagonism exerted by G. roseum against F. verticillioides is due, at least in part, to its detoxifying ability against FA., (© 2021. The Author(s) under exclusive licence to Society for Mycotoxin (Research Gesellschaft für Mykotoxinforschung e.V.) and Springer-Verlag GmbH Germany, part of Springer Nature.)