Lichens are demarcated as an association of two distinct and dissimilar components; the mycobiont (a fungus) and the phycobionts (an alga) forming a composite organism. The recent advancement in science has paid much attention on the biological roles of lichen secondary substances; which are attributed with a lot of biological activities, such as antibacterial, antifungal, antiviral, anti-inflammatory, anti-tumor, and antioxidant activities. The current research aims to unravel the fungicidal activity of lichen Heterodermia diademata, ethanolic extract (HDE) against prevalent superficial infection causing dermatophytes viz., Trichophyton rubrum, T. mentagrophytes, Microsporum gypseum and M. canis. The HDE was found active against M. gypseum and T. mentagrophytes with MIC; 1.68 and 1.98 mg/ml and mg/ml respectively. The dermatophytes were also analyzed by molecular phylogeny of chs-1 gene for variability in MIC and prediction of susceptible pathogens.