Polyomavirus infections are common and relatively benign in the general human population but can become pathogenic in immunosuppressed patients. Because most treatments for polyomavirusassociated diseases nonspecifically target DNA replication, existing treatments for polyomavirus infection possess undesirable side effects. However, all polyomaviruses express Large Tumor Antigen (T Ag), which is unique to this virus family and may serve as a therapeutic target. Previous screening of pyrimidinone–peptoid hybrid compounds identified MAL2-11B and a MAL2-11B tetrazole derivative as inhibitors of viral replication and T Ag ATPase activity (IC50 of ~20-50 μM. To improve upon this scaffold and to develop a structure–activity relationship for this new class of antiviral agents, several iterative series of MAL2-11B derivatives were synthesized. The replacement of a flexible methylene chain linker with a benzyl group or, alternatively, the addition of an ortho-methyl substituent on the biphenyl side chain in MAL2-11B yielded an IC50 of 50 μM, which retained antiviral activity. After combining both structural motifs, a new lead compound was identified that inhibited T Ag ATPase activity with an IC50 of 50 μM. We suggest that the knowledge gained from the structure–activity relationship and a further refinement cycle of the MAL2-11B scaffold will provide a specific, novel therapeutic treatment option for polyomavirus infections and their associated diseases.