Quaternary ammonium salts (QAS), as the surface active compounds, are widely used in medicine and industry. Their common application is responsible for the development of microbial resistance to QAS. To overcome, this issue novel surfactants, including gemini-type ones, were developed. These unique compounds are built of two hydrophilic and two hydrophobic parts. The double-head double-tail type of structure enhances their physicochemical properties (like surface activity) and biological activity and makes them a potential candidate for new drugs and disinfectants. Antimicrobial activity is mainly attributed to the biocidal action towards bacteria and fungi in their planktonic and biofilm forms, but the mode of action of gemini QAS is not yet fully understood. Moreover, gemini surfactants are of particular interest towards their application as gene carriers. Cationic charge of gemini QAS and their ability to form liposomes facilitate DNA compaction and transfection of the target cells. Multifunctional nature of gemini QAS is the reason of the long-standing research on mainly their structure-activity relationship.