Aspergillus clavatus poisoning is a neuromycotoxicosis of ruminants that occurs sporadically across the world after ingestion of infected feedstuffs. Although various toxic metabolites are synthesized by the fungus, it is not clear which specific or group of mycotoxins induces the syndrome. A. clavatus isolates were deposited in the culture collection of the Biosystematics Division, Plant Protection Research Institute, Agricultural Research Council during incidences of livestock poisoning (1988–2016). Six isolates were still viable and these plus three other South African isolates that were also previously deposited in the collection were positively identified as A. clavatus based on morphology and ß-tubulin sequence data. The cultures were screened for multiple mycotoxins using a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method. Twelve A. clavatus metabolites were detected. The concentrations of the tremorgenic mycotoxins (i.e., tryptoquivaline A and its related metabolites deoxytryptoquivaline A and deoxynortryptoquivaline) were higher than patulin and cytochalasin E. Livestock owners should not feed A. clavatus-infected material to ruminants as all the South African A. clavatus isolates synthesized the same compounds when cultured under similar conditions.