The present study is aimed to compare the in vitro antimicrobial and antioxidant activity of Acorus gramineus and Acorus calamus (Araceae). The antimicrobial activity of the two plants was evaluated disc diffusion and minimal inhibition concentration (MIC) method against six strains. The ethyl acetate fraction of rhizomes extract from A. gramineus exhibited more antimicrobial activity against the tested Gram-positive and Gram-negative bacteria as compared to the other tested fractions. The antioxidant activity was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay. The results showed that the half inhibitory concentration (IC50) of ethanol extract of A. gramineus rhizomes was the lowest value, it means the highest antioxidant activity, while total polyphenol content was very low. It showed that the antioxidant activity was not directly related to the polyphenol quantity. The results will be supported as a good information on the use of the two plants in the pharmaceutical and food industries.