The polytetrafluoroethylene (PTFE) serves as a dielectric layer for surface dielectric barrier discharge (SDBD) and the prepared plasma‐activated saline (PAS) exhibited strong bactericidal effects, however, the fluorides are inevitably generated and bring about side effects in biological application. In this study, the SDBD devices with the dielectric layers of quartz or ceramic compared with that with PTFE were utilized to explore the effective and fluoride‐free PAS using different working gases. The Quartz‐PAS prepared with air exhibited a relatively strong bactericidal effect without inducing the DNA‐protein crosslinks (DPCs) in Escherichia coli cells, while the Ceramic‐PAS exhibited a weak bactericidal effect and induced the DPCs formation. This study supplies guidance in the preparation and regulation of PAS for biomedical applications. [ABSTRACT FROM AUTHOR]