Sedimentary ancient DNA (sedaDNA) is an emerging field, increasingly being applied to the study of past aquatic ecosystems. However, several sedaDNA extraction methods from deep-ocean sediment matrices and sequencing library preparation have recently been developed, which may complexify results comparison and interpretations. We present a sedaDNA interlaboratory comparison study to assess the impact of extraction and library preparation protocols on metagenomic results. We applied three extraction protocols to four samples from two sediment cores from the Northern Antarctic Peninsula: (1) a ‘combined’ protocol using ethylenediaminetetraacetic acid (EDTA) and silica-in-solution to isolate DNA, (2) a high-guanidine protocol involving long cold centrifugation to remove polymerase chain reaction (PCR) inhibitors, and (3) a commercial kit, the DNeasy PowerSoil Pro Kit. We also compared two library preparation protocols, both optimised versions from Meyer and Kircher (2010). Using a blind approach relying on k-mer similarity assessment, greater variability was observed between individual samples than between protocols. An in-depth analysis of eukaryotic and (highly abundant) diatom community composition revealed that all protocols recovered eukaryotic sedaDNA, with minor differences between extraction and library protocols on sequence composition. However, the different DNA extraction and library preparations influenced the DNA read length, potentially resulting in selective targeting of organisms with variable sedaDNA preservation. This study highlights the importance of selection and standardisation of protocols to ensure reproducibility and comparability of past ecosystem reconstructions, particularly at lower taxonomic levels, e.g. diatoms. Although complete standardisation across research projects is challenging, this study shows that data remain reasonably comparable when processed consistently.