Fruits and vegetables are an essential part of a healthy diet, providing humans with vitamins, phytonutrients, and minerals. They are significantly vulnerable, however, to post-harvest diseases caused by numerous fungal and bacterial pathogens. These pathogens can cause significant quantitative and qualitative losses from harvest to consumption during the handling and storage processes. Chemical fungicides are commonly used but are likely to leave residues on the produce, rendering short shelf-life produce, such as berries, unsuitable for human consumption. Identifying eco-friendly methods to control post-harvest disease is, therefore, of utmost importance. The presence of antifungal constituents in the roots of Poncirus trifoliata extracts was detected by thin layer chromatography-based bioautography. The active constituents were isolated and identified by bioautography assay-guided fractionation using flash chromatography followed by spectroscopic techniques. In this study, xanthoxyletin, demethylsuberosin, dentatin, nordentatin, ponfolin, and clausarin were isolated from the root extracts. The antifungal activity of these compounds was moderate to weak compared to the commercial fungicide captan. This study reports the isolation and identification of natural compounds from Poncirus trifoliata that exhibited antifungal activity against Colletotrichum fragariae and Botrytis cinerea, two major post-harvest pathogens.