Tetralols 1 and 2, dihydroisocoumarins 3-6, and chromone 7 are natural compounds isolated from cultures of fungi, and their structures were determined by spectroscopic analyses. Compounds 1 and 2 from Nodulisporium sp. are novel tetralols, 1,2,3,4-tetrahydro-5-methoxynaphthalene-1,4-diol (nodulisporol) and 3,4-dihydro-4-hydroxy-8-methoxynaphthalen-1(2H)-one (nodulisporone), respectively. All isolated compounds selectively inhibited the activity of human DNA polymerase lambda (pol lambda), and compound 5 (3,5-dimethyl-8-methoxy-3,4-dihydroisocoumarin) was the strongest inhibitor of pol lambda in the tested compounds with an IC(50) value of 49 microM. New tetralols (1 and 2) are the third and second strongest inhibitors of pol lambda, but did not influence the activities of mammalian pols alpha to kappa, and showed no effect even on the activities of plant pols alpha and beta, prokaryotic pols, and other DNA metabolic enzymes such as calf terminal deoxynucleotidyl transferase, human immunodeficiency virus type-1 (HIV-1) reverse transcriptase, human telomerase, T7 RNA polymerase, and bovine deoxyribonuclease I. The structure-activity relationships of isolated compounds such as novel tetralols, dihydroisocoumarins, and chromone are discussed.