Herein we endow epoxidized natural rubber with antimicrobial activity by chemical modification. The rubber is firstly ring-opened by bromoacetic acid and then modified by antimicrobial group quaternary phosphonium salt. Using the hydrogen nuclear magnetic resonance spectroscopy (H-1 NMR) and the ICP atomic emission spectrometry (ICP-AES), the degree of epoxidation, the convert ratio of epoxy groups, and the phosphorus content of the products are measured, and then the effects of reaction time on the structure of products are investigated. The possible reaction mechanism and side reactions in ring-opening procedure are discussed through the comparison of data with different reaction time, and the ring-expansion reaction is supposed to be dominated in the side reactions. The thermal properties of modified rubbers and the structural factors behind are also investigated using differential scanning calorimeter and thermogravimetric analyzer. Antimicrobial activities of modified rubbers with different ring-opening reaction time toward Escherichia coli are studied, and the activity may increase with the content of quaternary phosphonium salt groups. This research may have the benefit for optimizing the structure of chemically modified antimicrobial rubbers.