Technical Abstract: Discovery of natural product fungicides is largely dependent upon the availability of high quality miniaturized antifungal bioassays. As part of a program to discover natural product-based fungicides, two sensitive assay systems were developed for evaluation of naturally occurring antifungal agents. Bioautography was used to identify the number of antifungal compounds in plant extracts, eliminate nuisance compounds, and characterize antifungal activity of pure compounds. Bioautography techniques were successful in evaluating antifungal compounds in strawberry, Artemisia douglasiana, Pimpinella species, and natural quinones. A second bioassay using a 96-well microtiter format was also developed to evaluate active fractions or pure compounds identified by bioautography. The 96-well plate assay has allowed us to study antifungal activity of sesquiterpene lactones, CAY-1, and compounds from Ruta graveolens. This coupled approach to natural product discovery combines the simple and visual nature of direct bioautography with the rapid, sensitive, and high throughput capabilities of a microtiter system. This will cover bioautography prescreens, microtiter assays (secondary assays), detached leaf assays for strawberry anthracnose (Colletotrichum spp.).