Saponaria sicula (Caryophyllaceae) is a perennial wild plant that grows exclusively on limestone cliffs on the Madonie Mts. and on the volcanic soil of Etna Mt., Sicily. This research aims to analyze the effect of pedo-climatic conditions on the leaf anatomy, micromorphology and phytochemistry of this species, sampled in the two above-cited sites. Moreover, the antioxidant and anti-inflammatory activities of leaf extracts were evaluated. Light microscopy analyses showed a higher amount of calcium oxalate druses in the leaves of Madonie plants (M). This is probably due to the higher CaCO3 and Ca richness in the soil with respect to that of Etna Mt. Toluidine blue O staining highlighted a positive reaction to polyphenols, mainly in the M leaves. These data agreed with the higher content of total phenols and flavonoids detected in the M hydroalcoholic extract (MHE) in comparison to that obtained from Etna plants (EHE). Furthermore, the evaluation of flavan-3-ols and proanthocyanidins content highlighted a low presence of polymeric tannins in both HEs. The phytochemical profile, evaluated using LC-DAD-ESI-MS analyses, revealed the presence of 64 compounds. Although, in both extracts, the most abundant compounds were the same (propelargonidine dimer, 1-synapoyl-2-feruloylgentiobiose and caffeoil glucose), statistically significant differences in terms of peak area percentage were recorded between MHE and EHE. According to the higher polyphenol content, MHE showed the highest biological activity, both from an antioxidant and anti-inflammatory point of view, with statistically significant differences in terms of half-inhibitory activity. In conclusion, this study experimentally demonstrates that pedo-climatic differences can affect the pharmacognostic features of this plant species.