In this study we used an analytical approach based on complementary techniques that targets all structural levels of collagen in leather to investigate how vegetable-tanned leather deteriorates during soil burial tests. For the first time, a group of deterioration markers specific to molecular, fibrillar and fibrous structure of collagen in leather was associated with the deterioration of buried leather. The application of the second order derivative of FTIR-ATR spectra analysis allowed us to detect loosening of collagen–tannin matrix, de-tanning and gelatin formation based on the behaviour of collagen and tannin spectral components (intensity variation and shifts). Collagen denaturation observed by DSC analysis and its thermo-oxidative behaviour measured by TG/DTG analysis, as well as the altered morphology of collagen (namely melt-like fibres and distorted fibrillar ultrastructure) imaged by SEM confirmed the FTIR-ATR analyis results. These analytical outcomes enabled us to understand the effect of leather hardening/cementing through soil mineral penetration into its fibrous structure and thus correctly interprete the higher-than-expected shrinkage temperatures and intervals determinatd by MHT method. Thus, MHT method proved to be suitable for a quick evaluation method that can direcly support the first conservation decision after excavation. The combination of FTIR-ATR, DSC, TG/DTG and SEM can be particularly useful to provide insights on the deterioration mechanism of archaeological leather and support best decision on its long-term preservation.