Protein disulfide isomerase (PDI) is an endoplasmic reticulum oxidoreductase/chaperone, and its dysregulation contributes to cancer progression, particularly glioblastoma. A high-throughput screen identified TC8026 as a PDI-active hit, and further optimization afforded a pyrrolo[2,3-d]pyrimidin-4-one series with up to 20-fold improved potency. Representative analogues (30w, 30z, 30aa, and 30ab) potently inhibited PDI, induced endoplasmic reticulum stress-mediated apoptosis in glioblastoma cells, and 30z significantly suppressed tumor growth in a U251 xenograft model. Mechanistic studies revealed a previously unrecognized allosteric-covalent binding mode. The inhibitors initially engage an allosteric pocket within the b′ domain involving residues H256 and F304, thereby perturbing the substrate-binding interface and inducing conformational changes that expose the noncatalytic cysteine C312 for covalent capture. This b′-directed allosteric covalency, distinct from conventional catalytic cysteine modification, confers enhanced selectivity within the PDI family. These findings define a novel allosteric-covalent chemotype of PDI inhibitors with a unique binding mechanism and promising antiglioblastoma potential.