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Pilea microphylla (P. microphylla), small shrubs that thrive in shaded environments, which belongs to the family Urticaceae. Currently this plant gained attention due to the medical properties it contains. The main objective of this study is to investigate the enzyme inhibitory, antioxidant and phytotoxic properties of Pilea microphylla. For that methanolic extracts were prepared from the P. microphylla and their inhibitory activities against key enzymes mainly α-amylase, acetylcholinesterase, and lipase, antioxidant potential, Antifungal and phytotoxic effects were evaluated using appropriate assays. The findings revealed that the methanolic extract did not demonstrate antifungal properties, cytotoxicity, or α-amylase inhibitory activity. The IC50 values obtained for the antioxidant, acetylcholinesterase inhibitory, and lipase inhibitory assays were 46.69 mg L-1, 132.531 mg L-1, and 25.148 mg L-1, respectively. Notably, the extract exhibited significant inhibitory activity on root growth in the test plant (50.12%), but its effect on shoot growth was less pronounced (8.84%). In conclusion, the study highlighted the bioactivity of P. microphylla methanolic extracts. While the extract did not exhibit antifungal or cytotoxic properties, it demonstrated moderate antioxidant activity and inhibitory effects on acetylcholinesterase and lipase enzymes. Additionally, the extract showed phytotoxic activity by inhibiting root growth. Further research and analysis of P. microphylla and its extracts may contribute to our understanding of its potential applications in various fields.