Extensive studies on the antimicrobial activity of terpene-based substances, which are the main components of essential oils, are continuously underway. And some hydrocarbons constituting antimicrobial substances have been reported to exhibit antimicrobial activity. This study confirmed the production of isoprene, the most basic constituent hydrocarbon of terpene, by Rhodobacter sphaeroides, and investigated the antimicrobial activity of isoprene and its mechanism. In dark-aerobic conditions, R. sphaeroides shows the highest growth-coupled isoprene production. We developed an air-sharing culture system that can be cultured by sharing the atmosphere, and we evaluated the antimicrobial activity of the isoprene released from R. sphaeroides on two Gram-negative bacteria and two Gram-positive bacteria. As a result, the isoprene released from R. sphaeroides showed antimicrobial activity against all evaluated strains, especially against Gram-positive bacteria than Gram-negative bacteria. In addition, the microstructure of the bacteria was evaluated via Field Emission Scanning Electron Microscopy (FE-SEM). The FE-SEM images showed that isoprene has antimicrobial activity mechanism that causes cell death by acting on the cell wall or the extracellular membrane.