Piper retrofractum is a medicinal plant. Bioactive compounds from its fruits have been recognized and used in food, cosmetics, and medicine. Insecticidal and antimicrobial compounds in this plant have also been reported, but there have been no relevant studies on their phytotoxic activity. Therefore, this study aimed at the evaluation of phytotoxic activity of an aqueous methanol extract of the fruits of P. retrofractum and the activity of the isolated compounds on seedling growth of test plants. The result showed that the fruit extracts were phytotoxic to cress, lettuce, alfalfa, barnyard grass, Italian ryegrass, and jungle rice seedlings. A significant reduction in growth of all test seedlings was observed for the extract at 1 mg dry weight equivalent extract/mL. The concentrations required for 50% growth inhibition (I50) of the extract on all test seedlings ranged from 0.3 to 14 mg dry weight equivalent extract/mL. The extract was then subjected to bioassay-guided fractionations, and seven compounds, 3-phenylpropanoic acid (1), (2E,4E)-methyl piperate (2), (2E,4Z)-methyl piperate (3), piperlonguminine (4), dihydropiperine (5), isochavicine (6), and piperine (7) were isolated. Those compounds inhibited seedling growth of cress and barnyard grass and the inhibition increased with increasing concentrations of the compounds. Compounds (6), (7), and (4) inhibited shoot and root growth of cress at concentrations greater than 30 ?M, with I50 values ranging from 11 to 18, 23 to 35, and 25 to 10 ?M, respectively. Compounds (7) and (5) inhibited shoot and root growth of barnyard grass at concentrations greater than 100 and 300 ?M, with I50 values ranging from 238 to 37 and 487 to 131 ?M, respectively. These results suggest that the compounds (1-7), especially (4-7), may be responsible for the phytotoxic activity of the fruit extract of P. retrofractum.