Protein disulfide isomerase (PDI) is responsible for nascent protein folding in the endoplasmic reticulum (ER) and is critical for glioblastoma survival. To improve the potency of lead PDI inhibitor BAP2 (( E)-3-(3-(4-hydroxyphenyl)-3-oxoprop-1-en-1-yl)benzonitrile), we designed and synthesized 67 analogues. We determined that PDI inhibition relied on the A ring hydroxyl group of the chalcone scaffold and cLogP increase in the sulfonamide chain improved potency. Docking studies revealed that BAP2 and analogues bind to His256 in the b' domain of PDI, and mutation of His256 to Ala abolishes BAP2 analogue activity. BAP2 and optimized analogue 59 have modest thiol reactivity; however, we propose that PDI inhibition by BAP2 analogues depends on the b' domain. Importantly, analogues inhibit glioblastoma cell growth, induce ER stress, increase expression of G2M checkpoint proteins, and reduce expression of DNA repair proteins. Cumulatively, our results support inhibition of PDI as a novel strategy to treat glioblastoma.