Plants can stimulate bacterial nitrogen (N) removal by secretion of root exudates that may serve as carbon sources as well as non-nutrient signals for denitrification. However, there is a lack of knowledge about the specific non-nutrient compounds involved in this stimulation. Here, we use a continuous root exudate-trapping system in two common aquatic duckweed species, Spirodela polyrrhiza (HZ1) and Lemna minor (WX3), under natural and aseptic conditions. An activity-guided bioassay using denitrifying bacterium Pseudomonas fluorescens showed that crude root exudates of the two species strongly enhanced the nitrogen-removal efficiency (NRE) of P. fluorescens (P < 0.05) under both conditions. Water-insoluble fractions (F) obtained under natural conditions stimulated NRE to a significant extent, promoting rates by about 30%. Among acidic, neutral and basic fractions, a pronounced stimulatory effect was also observed for the neutral fractions from HZ1 and WX3 under both conditions, whereas the acidic fractions from WX3 displayed an inhibitory effect. Analysis of the active fractions using gas chromatography/mass spectrometry (GC/MS) revealed that duckweed released fatty acid methyl esters and fatty acid amides, specifically: methyl hexadecanoate, methyl (Z)-7-hexadecenoate, methyl dodecanoate, methyl-12-hydroxystearate, oleamide, and erucamide. Methyl (Z)-7-hexadecenoate and erucamide emerged as the effective N-removal stimulants (maximum stimulation of 25.9 and 33.4%, respectively), while none of the other tested compounds showed stimulatory effects. These findings provide the first evidence for a function of fatty acid methyl esters and fatty acid amides in stimulating N removal of denitrifying bacteria, affording insight into the "crosstalk" between aquatic plants and bacteria in the rhizosphere.