Owing to the rise in antimicrobial and chemotherapeutic drug resistance, there is a desperate need to formulate newer as well as more effective agents. With this perspective, here we outline the synthesis of two novel gemini surfactants with different substitutions at the nitrogen atom of the benzimidazolium ring. Both the compounds induced significant reductions in Candida growth in various yeast strains. The reduction in Candida growth seemed likely through the reduction in ergosterol biosynthesis: a sterol constituent of yeast cell membranes. Different concentrations of both compounds were used to determine the cellular ergosterol content which indicates an important disordering of the ergosterol biosynthetic pathway. Cytotoxic studies were carried out using HEK 293 (human embryonic-kidney cells) and Galleria mellonella larvae (an in vivo model of antimicrobial studies). Administration of both the compounds to G. mellonella larvae diseased by the yeast Candida albicans resulted in increased survival indicating their in vivo activity.