Triiodide resin (TR) was used to generate axenic cultures of microalgae by employing the antibacterial capability of triiodide. A Nannochloropsis gaditana culture contaminated with bacteria was passed through a column filled with TR using the gravity flow. Based on analyses of flow cytometry and vital staining using a fluorescent dye SYTOX Green, three cycles of TR treatments remarkably reduced the number of viable bacteria but had little effects on the microalgae. This novel approach is a simple, rapid, and cost-effective method that can be used to isolate axenic cultures of microalgae.