Corynetoxins are a group of toxins associated with annual ryegrass toxicity (ARGT) in animals grazing annual ryegrass parasitised by the nematode Anguina agrostis and bacterium Corynebacterium rathayi. Previous studies explored plant, nematode, or bacterial origins of the toxins, and recent identification of corynetoxins as members of the tunicamycin group of antibiotics suggested a bacterial origin. This study investigated production of corynetoxins (CT) by C. rathayi in liquid cultures. C. rathayi was grown in modified Rado and Heskett medium, cells and supernatant were extracted, and CT was analyzed via HPLC, UV spectroscopy, and biological assays (antibacterial activity against Bacillus subtilis and antiglycosylation activity). CT was detected in cell extracts (not in supernatant) and showed chromatographic, spectral, and biological properties closely matching authentic CT from parasitised seedheads. While chemical confirmation was limited by low yields, the consistent properties provide strong presumptive evidence that CT is produced by C. rathayi in liquid culture, confirming a bacterial origin.