Introduction: Cover crops, like black oat, suppress weeds in multiple ways, e.g. by resource competition and/or root exudates, but very little is known about their root exudate composition. Methods: Root exudates from black oat interacting with intra- (black oat) and interspecific (redroot pigweed and blackgrass) neighbors were collected using split-root systems and analyzed with mass spectrometry. Changes in black oat root exudation patterns and root morphology were assessed. Moreover, collected exudates were applied to weeds to observe the effect on weed root traits. Results: Redroot pigweed root traits declined when grown with black oat, whereas blackgrass showed the opposite trend. Upon black oat root exudate application, pronounced effects on redroot pigweed root traits were observed, whereas there was no reaction in blackgrass. The proportionally more accumulated compounds in root exudates in response to neighbors were primarily organic oxygen compounds, most annotated as carbohydrates. In the presence of redroot pigweed, root exudation increased, with sugar sulphates and potential glycosylamines identified as priority compounds. Discussion: This study provides the first characterization of black oat root exudates and demonstrates the varying patterns of black oat root exudation when interacting with different neighbors and their influence on root traits.