The utilisation of massively parallel sequencing (MPS) in forensic DNA analysis is on the rise, driven by the expansion of targeted MPS panels in the market and the introduction of forensic investigative genetic genealogy. The MPS library preparation process, integral to both whole-genome sequencing (WGS) and targeted MPS panel data generation, is largely based on converting double-stranded DNA (dsDNA) into sequencing libraries. In the current study, we examined the effect of seven routinely used forensic DNA extraction methods on the strandedness (single-stranded or double-stranded) and the fragment size of the DNA extracted from buccal swab, blood, bone and tooth samples. Our findings reveal a variation in the proportion of dsDNA and single-stranded DNA (ssDNA), with the phenol-chloroform and silica column-based extraction methods tested predominantly yielding dsDNA, while the tested Chelex and magnetic bead-based extraction methods predominantly yielded ssDNA. Additionally, fragment size analysis showed that high molecular weight dsDNA was recovered from buccal swab samples with all of the extraction methods except Chelex, which yielded relatively short dsDNA fragments. DNA extracted from tooth samples with tested magnetic bead-based extraction methods resulted in longer dsDNA fragments compared to the silica column-based extraction protocol.