Guidelines identifying appropriate DNA extraction methods for both museum and modern biological samples are scarce or non-existent for mammalian species. Yet, obtaining large-scale genetic material collections are vital for conservation and management purposes. In this study, we evaluated five protocols making use of either spin-column, organic solvents, or magnetic bead-based methods for DNA extraction on skin samples from both modern, traffic-killed (n = 10) and museum (n = 10) samples of European hedgehogs, Ericaneus europaeus. We showed that phenol-chloroform or silica column (NucleoSpin Tissue) protocols yielded the highest amount of DNA with satisfactory purity compared with magnetic bead-based protocols, especially for museum samples. Furthermore, extractions using the silica column protocol appeared to produce longer DNA fragments on average than the other methods tested. Our investigation demonstrates that both commercial extraction kits and phenol-chloroform protocol retrieve acceptable DNA concentrations for downstream processes, from degraded remnants of traffic-killed and museum samples of mammalian specimens. Although all the tested methods could be applied depending on the research questions and laboratory conditions, commercial extraction kits may be preferred due to their effectiveness, safety and the higher quality of the DNA extractions.