In this study, Peperomia pellucida was subjected to the determination of biological activity and isolation of chemical constituents. Two samples of this plant were used, which were the air-dried and freeze-dried. The sequential maceration of the plant utilized three different solvents with increasing polarities, namely dichloromethane, ethyl acetate and methanol. The Thin Layer Chromatography (TLC) profile showed there was no significant different in the chemical constituents in both samples. All six crude extracts displayed in vitro antimicrobial activity on Pseudomonas aeruginosa, with minimum inhibition zone 9.0±1.7 mm and no activity on 7 other type of bacteria (Escherichia coli, Klebsiella pneumonia, Salmonella sp., Bacillus subtilis, Micrococcus sp., Staphylococcus aureus and Streptococcus uberis). In addition, antioxidant activity of all of the crude extracts was determined using DPPH (2,2-diphenyl-picryhdrazyl) radical scavenging assay. Among the extracts methanol extract of freeze dried sample showed the highest antioxidant activity with IC50 value of 2.45±0.20 mg/ml. The result also showed that the free radical scavenging activity was proportional to the concentrations of samples. The isolation of compound from dichloromethane extracts was performed by using various chromatographic techniques such as dry column vacuum chromatography, chromatotron and preparative thin layer chromatography techniques and produce six single compounds, with three of them were successfully characterized as dillapiole, caryophyllene oxide and stigmasterol. The structures of the compounds were determined using IR, UV, EIMS and NMR spectroscopies.