A library of 500 Australian marine invertebrate extracts was screened for anti-prion activity using a yeast-based assay, and this resulted in an extract from the ascidian <i>Polycarpa procera</i> showing potent activity. Purification of this extract led to the isolation of six new butenolide metabolites, the procerolides <b>1</b>-<b>4</b> and two related diphenylpropanones, the procerones <b>5</b> and <b>6</b>, as the bioactive components. The structures of <b>1</b>-<b>6</b> were elucidated from the analysis of 1D/2D NMR and MS data, and their absolute configurations determined from comparison of experimental and computed ECD data. Compounds <b>1</b>-<b>6</b> were tested for anti-prion activity in a yeast-based assay, and <b>1</b> and <b>5</b> displayed potent bioactivity (EC<sub>50</sub> of 23 and 29 μM, respectively) comparable to the potently active anti-prion compound guanabenz. The procerolides and procerones are the first anti-prion compounds to be reported from ascidians, indicating that ascidians may be an untapped source of new lead anti-prion compounds.