Agricultural run-off sedimentation reservoirs are an emerging aquatic system of critical importance to plant biosecurity, water and environmental sustainability. Oomycete pathogens such as Phytophthora and Pythium species in irrigation water have been demonstrated to pose significant risks to ornamental crops, but little is known about their diversity and populations in sediments of agricultural irrigation systems. This study investigated the oomycete communities including Phytophthora (Ph.), Phytopythium (Pp.) and Pythium (Py.) species in sediments at various depths of an agricultural run-off sedimentation reservoir in Virginia during the winters of 2011 and 2015. The recovery of these oomycetes declined sharply with sediment depth from surface to 0·8 m and none was recovered from sediments deeper than 1·4 m. A total of 47 oomycete species were recovered, with all four species of Phytophthora and five of Phytopythium exclusively from the surface. Recovered species included many important plant pathogens such as Ph. nicotianae, Ph. pini, Ph. tropicalis, Pp. vexans, Py. irregulare and Py. monospermum. These results underline the importance of decontaminating sediments excavated from top layers (0–1·4 m) of the sedimentation reservoir before reuse in plant production.