Biological contamination poses a critical challenge to microalgae cultivation, yet effective and microalga-safe control agents remain limited. In this study, 1,8-cineole, a major component of eucalyptus oil, was evaluated as an insecticide to investigate its effects on biological contaminants Euplotes vannus, Brachionus plicatilis, and Tigriopus japonicus during the cultivation of microalage Synechococcus sp., Chlorella sp., and Isochrysis sp., respectively. Results indicated that 1,8-cineole significantly reduced the abundance of the three biological contaminants while causing a negligible effect on microalgal abundance and photosynthetic efficiency. And exposure to 1,8-cineole altered the antioxidant enzyme activities and antioxidant contents of the biological contaminants, whereas the protein and lipid contents of the microalgae remained virtually unchanged. Transcriptome sequencing revealed that the impact of 1,8-cineole on the three biological contaminants primarily involved accelerated energy metabolism, altered cellular structures, and regulated growth and development processes. This research provides an innovative biofouling solution, aiding sustainable microalgae production.