Background: Phyllanthus niruri, in Indonesia, is known as “Meniran” has a long history of use in ethnic or traditional medicine worldwide, mainly as an antihypertensive agent. Objective: The present study was designed to isolate and identify active compounds with angiotensin-converting enzyme (ACE) inhibition activity from P. niruri herb and confirm the mechanism of action, affinity, and domain specificity interactions of the isolated compounds. Materials and Methods: Some fractions of P. niruri methanolic extract were subjected to column chromatography and preparative thin-layer chromatography to get active compounds. Structural elucidation was determined via spectroscopic methods. ACE inhibition activity was measured using hippuryl-L-histidyl-L-leucine as a substrate in vitro assay. Furthermore, confirmation of the mechanism of action, affinity, and domain specificity interaction of the isolated compounds on ACE complex macromolecule (protein database id: 1O86) was performed by in silico molecular docking studies. Results: In this work, four active compounds were isolated from aerial part of P. niruri, including hypophyllantin (50% inhibition concentration [IC50] = 0.180 μg/mL), phyllantin (IC50 = 0.140 μg/mL), methyl gallate (IC50 = 0.015 μg/mL), and quercetin 3-O-β-D-glucopyranosyl-(1'''-6'')-α-rhamnoside (IC50 = 0.086 μg/mL). In silico molecular docking method emphasizes ligand-residue interactions, thereby predicting the inhibitory activity of these compounds. After docking to an ACE complex macromolecule, quercetin 3-O-β-D-glucopyranosyl-(1'''-6'')-α-rhamnoside obtained more interactions than lisinopril. Conclusion: The results were obtained from in silico and in vitro experiments and confirm the potential active compound is an ACE inhibitor and a new antihypertensive agent.