The fragmented ecosystems along the Niagara Escarpment World Biosphere Reserve provide important habitats for biota including lichens. Nonetheless, the Reserve is disturbed by dense human populations and associated air pollution. Here we investigated patterns of lichen diversity within urban and rural sites at three different locations (Niagara, Hamilton, and Owen Sound) along the Niagara Escarpment in Ontario, Canada. Our results indicate that both lichen species richness and community composition are negatively correlated with increasing human population density and air pollution. However, our quantitative analysis of community composition using canonical correspondence analysis (CCA) indicates that human population density and air pollution is more independent than might be assumed. The CCA analysis suggests that the strongest environmental gradient (CCA1) associated with lichen community composition includes regional pollution load and climatic variables; the second gradient (CCA2) is associated with local pollution load and human population density factors. These results increase the knowledge of lichen biodiversity for the Niagara Escarpment and urban and rural fragmented ecosystems as well as along gradients of human population density and air pollution; they suggest a differential influence of regional and local pollution loads and population density factors. This study provides baseline knowledge for further research and conservation initiatives along the Niagara Escarpment World Biosphere Reserve.