In the present study, quantitative analysis of usnic acid and the selected elements (Ag, Ca, Cd, Cr, Cu, Mg, Mn, Ni, Pb, Zn) in samples of Cladonia mitis thalli collected along a North-Central Europe transect, was performed. Additionally, we analysed geographical and environmental factors that could potentially influence the levels of the analytes. With the constructed partial least square (PLS) chemometric model we aimed to establish the relationships between the analysed factors. Our study is the first to provide comparative quantitative data on usnic acid in C. mitis. We proved this lichen species is a rich source of usnic acid (4.52 ± 0.54-21.58 ± 2.23 mg/g dry wt). Significant differences in usnic acid content between samples collected in the open and forest areas were noted, with the predominance of the former (mean 17.4 ± 2.8 and 8.8 ± 2.3 mg/g dry wt, respectively). Our results supported the hypothesis on the relationship between usnic acid and selected heavy metals content in lichens, as the positive correlation of the compound was noted with Pb. Our PLS model indicated also a positive correlation between usnic acid and Cr content, which was reported for the first time.