Objective: Gamma irradiation is an effective technique can be used to reduce contaminants in herbal products. Peperomia pellucida (L.) Kunth belongs to Piperaceae family has some biological activity, such as antioxidant and ACE inhibitor. The aimed of this research was to determine the effects of gamma irradiation on microbiological, phytochemical content, antioxidant and ACE inhibition activity. Methods: Sample was irradiated at a various dose of 0, 2.5, 5, 7.5 and 10 kGy. Microbiological was determined as total aerobic plate count (TAPC), and total yeast and mold (TYC), total phenolic content (TPC), total flavonoid content (TFC), antioxidant and ACE inhibition activity were investigated by various in vitro colorimetric methods. Results: Irradiation dose of 10 kGy reduced TAPC and TYC. Irradiation dose of 10 kGy slightly increased TPC, antioxidant, and ACE inhibition activity (p<0.05) but reduced TFC significantly (p<0,05) and caused degradation of flavonoids in this plant. Correlation analysis showed that phenolic compounds have a role as antioxidant and ACE inhibitor. Conclusion: gamma irradiation was found to be sufficient to ensure microbiological safety, increase TPC, the antioxidant and ACE inhibitory activity but caused degradation of flavonoids on Peperomia pellucida (L.) Kunth.This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.