Tetramic acids constitute a large class of natural products isolated from a variety of different fungal and bacterial species. While the presence of the distinctive 2,4-pyrrolidinedione ring system defines this class of compounds, these compounds are widely diverse both structurally and in the biological activities that they display. Equisetin-like compounds are tetramic acids that have been shown to be growth inhibitory towards bacteria, fungi, yeasts and mammalian cell lines; however, the mechanisms inhibiting prokaryotic and eukaryotic cell growth have not been fully explained. Here we report the isolation and biological characterisation of a novel equisetin-like tetramic acid named tetramic acid-289 (TA-289) produced by a Fusarium sp. fungus. This compound displayed pH- and carbon source-dependent cytotoxic effects in Saccharomyces cerevisiae and caused an irreversible cell cycle block via a microtubule independent mechanism. To fully elucidate a mechanism, we used an unbiased approach employing chemogenomic profiling of the yeast deletion library and demonstrated that TA-289 hypersensitive deletion strains are also sensitive to oxidants, respiratory inhibitors and have abnormal mitochondrial morphology. In support of the hypothesis that TA-289 perturbs mitochondrial function, we demonstrated the ability of this compound to generate reactive oxygen species only during fermentative growth, an effect reliant on an intact electron transport chain. In addition, mitochondrial morphological defects were detected upon exposure to TA-289 independent of the increase in oxidative stress. The generation of reactive oxygen species was not the sole cause of cell death by TA-289, as only partial amelioration of cell death was achieved by the deletion of genes encoding components of the electron transport chain, despite these deletions causing attenuation of the magnitude of oxidative stress. We propose that TA-289 induces cell death via the direct inhibition of a mitochondrially localised target or targets, and that the mitochondrial morphology defect and ROS production observed in this study is a direct consequence of the induction of cell death. This study highlights the complex interplay between mitochondrial function, cell death and the generation of reactive oxygen species when elucidating the mode-of-action of compounds that cause oxidative stress and cell death, and further deepens the mystery surrounding the molecular basis of the activity of equisetin-like compounds.