In the present study, the n-hexane extract of Myristica fragrans (MF) seeds, acetone-insoluble part of the n-hexane extract (AIMF) and trimyristin (TM) were assessed for their anxiogenic activity. The MF (10 and 30 mg/kg), AIMF (30, 100, and 300 mg/kg), and TM (10, 30, and 100 mg/kg) administered intraperitoneally exhibited anxiogenic activity in elevated plus-maze (EPM) paradigm. The open-field test and hole-board test were also used to assess anxiogenic activity of AIMF and TM. In the EPM test, MF, AIMF, and TM decreased the time spent by mice in the open arm and the entries in the open arm. Further, the effect of diazepam (1 mg/kg i.p.), serotonin 5-HT3 receptor antagonist, ondansetron (1 mg/kg i.p.), and 5-HT1A receptor agonist, buspirone (1 mg/kg i.p.), on the occupancy in open arm and entries in open arm was significantly reduced by TM. In the open-field test, AIMF as well as TM reduced the number of rearing and locomotion. Both TM and AIMF reduced the number of head pock in the hole-board test. Inhibition of anxiolytic activity of ondansetron (5-HT3 receptor antagonist), buspirone (5-HT1A receptor agonist), and diazepam [acting on gamma-aminobutyric acid (GABAA) receptor] suggests a nonspecific anxiogenic activity of TM and also a link between 5-HT and GABA systems in the anxiogenic activity of TM.