We report the synthesis and cytotoxicity evaluation of a completely new class of cationic carbosilane dendrimers functionalized with several different phosphonium peripheral groups and an ammonium functionalised one as a reference. The carbosilane dendrimers with NMe₃, PMe₃, P(Et₂)₂(CH₂)₃OH, PBu₃, P(C₆H₄-OMe)₃ and P(Ph)₃ peripheral substituents were synthesized, thoroughly characterized and modelled by computer simulations. The cytotoxicities of the dendrimers were investigated in vitro on three model cell lines (B14, BRL and NRK cells) by MTT and CV assay methods. Generally, the cytotoxicities of PMe₃ carbosilane dendrimers were similar or slightly lower when compared with NMe₃ dendrimers. The substitution of methyl groups in PMe₃ carbosilane dendrimers with more hydrophobic and bulky alkyl substituents (PBu₃ and P(Et₂)₂(CH₂)₃OH dendrimers) resulted in an increase of cytotoxicity. The P(C₆H₄-OMe)₃ dendrimer showed exceptionally low cytotoxicity across all cell lines or assay methods used. Generally, phosphonium carbosilane dendrimers could represent a valuable alternative to ammonium ones in gene therapy applications due to comparable or lower cytotoxicities, the presence of positive charge for nucleic acid electrostatic binding and in the cases of P(C₆H₄-OMe)₃ and P(Ph)₃ dendrimers high potential of mitochondrial targeting.