Thamnolia vermicularis (Tv) and Thamnolia subuliformis (Ts) are two species from the same habitat with such similar external morphological characteristics that researchers often confuse the two in their scientific work and do not distinguish between them. This study applies three different methods to distinguish them. The diversity of endophytic fungi was also compared and their antibacterial activity in vitro was evaluated. The results show that all three methods can distinguish Tv and Ts, and can be used to cross-validate each other. The ultraviolet fluorescence method and the chemical colour change method are simpler strategies, while thin-layer chromatography is relatively complicated but can more clearly distinguish the chemical composition of the two species. In the analysis of the endophytic fungi community structure of Tv and Ts, it was found that the diversity of endophytic fungi in Tv was more abundant and had wider antibacterial activity and better inhibitory activity against gram-positive bacteria in vitro. Keywords: Lichen; Ultraviolet fluorescence; Chemical chromatography; Thin-layer chromatography; Antibacterial activity; ITS sequencing.