By using a homology-based bioinformatics approach, a structural model of the vaccinia virus (VV) I7L proteinase was developed. A unique chemical library of ∼51,000 compounds was computationally queried to identify potential active site inhibitors. The resulting biased subset of compounds was assayed for both toxicity and the ability to inhibit the growth of VV in tissue culture cells. A family of chemotypically related compounds was found which exhibits selective activity against orthopoxviruses, inhibiting VV with 50% inhibitory concentrations of 3 to 12 μM. These compounds exhibited no significant cytotoxicity in the four cell lines tested and did not inhibit the growth of other organisms such as Saccharomyces cerevisiae, Pseudomonas aeruginosa, adenovirus, or encephalomyocarditis virus. Phenotypic analyses of virus-infected cells were conducted in the presence of active compounds to verify that the correct biochemical step (I7L-mediated core protein processing) was being inhibited. Electron microscopy of compound-treated VV-infected cells indicated a block in morphogenesis. Compound-resistant viruses were generated and resistance was mapped to the I7L open reading frame. Transient expression with the mutant I7L gene rescued the ability of wild-type virus to replicate in the presence of compound, indicating that this is the only gene necessary for resistance. This novel class of inhibitors has potential for development as an efficient antiviral drug against pathogenic orthopoxviruses, including smallpox.