Cationic surfactants are widely used as antimicrobial agents in various fields. However, the widespread use of cationic surfactants has caused huge accumulation in environment, triggering the emergence of bacterial resistance. Besides, owing to the strong cytotoxicity, most of the cationic surfactants are limited in practical applications. Therefore, constant attention has been paid on how to rationally design the surfactants for achieving highly efficient antimicrobial activity at lower doses but simultaneously presenting low toxic side effects. In this review, we mainly focus on recent advances in the key structural determinants of antibacterial activity and cytotoxicity of cationic surfactants, as well as the design strategies of effective antimicrobial surfactants with low cytotoxicity.