N-Acylethanolamines (NAEs), although are ubiquitous in plants and animals the occurrence of endocannabinoid ligands and the corresponding cannabinoid receptors was limited to mammals. Interestingly, bryophytes, unlike seed plants possess arachidonic acid (AA, 20:4) and eicosapentaenoic acid (EPA, 20:5), which are fatty acid precursors for endocannabinoid ligands. Here, we show that the moss Physcomitrella patens contains ~24 and 7 % of AA and EPA, respectively. Using selective lipidomic profiling, we identified polyunsaturated NAEs, including N-arachidonoyl ethanolamide (anandamide/AEA/NAE 20:4) and N-eicosapentaenoyl ethanolamide (EPEA) and also their corresponding N-acyl-phosphatidylethanolamine (NAPE) precursors in various developmental stages of Physcomitrella. Quantification of various NAPE and NAE species indicated the abundance of unsaturated species over saturated. In all haploid developmental stages analyzed, NAE 20:4 levels contributed to ~ 30 % (~ 26 ng mg-1 lipid) of the total NAE while NAE 20:5 remained as a minor component (~ 5 %; ~ 4.5 ng mg-1 lipid). Exogenous application of AEA, EPEA and their corresponding fatty acid precursors (AA and EPA, respectively) inhibited the growth of gametophytes and protonemata in a dose-dependent manner. AEA has shown the exclusive effect on the F-actin dynamics at the apex of protonemal cells, which was similar to the effect of abscisic acid (ABA) on protonemal growth inhibition. Additionally, we identified moss ortholog for NAPE-hydrolyzing phospholipase D (NAPE-PLD) enzyme that was responsive to exogenous ABA. Putative PpNAPE-PLD was expressed in E. coli for further characterization. Our data demonstrate the occurrence of evolutionarily conserved NAE metabolic pathway in the moss, with the occurrence of AEA and EPEA.