This study is a continuation and extension of our previous study in which we synthesized seven novel eugenol tosylate congeners (ETC-1 to ETC-7) from a natural compound eugenol and checked their antifungal activity against different isolates of Candida albicans. All these ETCs showed potent antifungal activity to varying degrees. In this study, the aim is to evaluate the effect of most active compounds (ETC-5, ETC-6 and ETC-7) on ergosterol biosynthesis pathway and cellular viability in C. albicans by applying combined approach of in silico and in vitro methodologies. In silico studies were done through all atom molecular mechanics approach and free binding energy estimations, and in vitro study was done by estimating total intracellular sterol content and effect on expression of ERG11 gene. Furthermore, effect on cell viability by these compounds was also tested. Our results demonstrated that these ETCs target ergosterol biosynthesis pathway in C. albicans by inhibiting the lanosterol 14-α demethylase enzyme and also downregulates expression of its related gene ERG11. Furthermore, these ETCs exhibit potent fungicidal effect in cell viability assay, thus overall results advocating the claim that these tosylates have potential to be taken to next level of antifungal drug development.