Gemini surfactants consisting of two melamine scaffolds connected by a n-hexyl linker and functionalized with a 1-propylammonium polar head and a lipophilic chain having variable carbon length (from C8 to C16) were synthesized. These were then used successfully for the transfection of A549, U87 MG, and Bristol 8 cell lines with maxGFP expressing plasmid. The transfection protocol was optimized appropriately (confluence, reagent/pcDNA ratio, compaction time, and transfection time) for each cell line. Under optimized conditions, the C12 and C14 melamine gemini surfactants showed little toxicity and remarkable transfection efficiency, superior to the gold-standard Lipofectamine 2000. These reagents were also able to efficiently transfect primary DRG neurons, which are notoriously difficult to transfect. The presence of serum completely inhibited the transfection capacity of these reagents. Owing to their ready availability, straightforward synthesis, high chemical stability (even in solution), ease of use (no formulation is required), improved transfection ability, and low toxicity, melamine-based gemini surfactants are very promising reagents for cellular DNA transfection.