We have identified a series of novel inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase by randomly screening natural product extracts using an in vitro biochemical assay designed to identify inhibitors of integrase-catalysed strand transfer. Equisetin recovered from the fungus Fusarium heterosporum and a novel enantiomeric homologue of equisetin from Phoma sp. were isolated as inhibitors of HIV-1 integrase in vitro. Two additional analogues, a novel decalin derivative, integric acid, and oteromycin were also discovered to be inhibitors of integrase. Equisetin and related compounds inhibit 3' end-processing and strand transfer as well as disintegration catalysed by either the full-length enzyme or the truncated integrase core domain (amino acids 50-212). These compounds also inhibit strand transfer reactions catalysed by stable complexes assembled in vitro and integration reactions catalysed by pre-integration complexes isolated from HIV-1-infected cells. The compounds described in this report are structurally novel and mechanistically distinct from many previously described inhibitors of HIV-1 integrase. These results demonstrate the utility of using an appropriately configured assay to identify compounds that are effective post-assembly and the potential of isolating novel integrase inhibitors from complex natural product extracts.