The emergence of new data on the association between the composition of the intestinal microbiota and various human diseases has generated increasing interest in microbiome research. In this context, selection of the DNA extraction method represents a critical stage in the design of the experiment, significantly affecting the reliability and reproducibility of results. This study presents a comparative analysis of 12 DNA extraction methods, including nine commercial kits and three laboratory protocols. We evaluated the taxonomic representation, including Gram-positive (Lactobacillaceae, Coprococcus spp., Streptococcus sp., Clostridium leptum) and Gram-negative bacteria (Enterobacteriaceae, Akkermansia muciniphila, Fusobacterium nucleatum, Bacteroides fragilis). The extraction efficiency was assessed by the DNA yield, expressed in GE/pL of eluate or in GE/g of feces, as well as by the frequency of low-abundance taxa loss. Clustering of the methods according to the type of lysis was demonstrated: mechanical lysis provided stable and high DNA yields, particularly for Gram-positive bacteria, while chemical and enzymatic methods showed lower efficiency. We determined that the lysis type and pre-processing of intact fecal samples are the key factors affecting the DNA extraction efficiency and preservation of the native taxonomic profile. The best results were demonstrated by the QIAamp® PowerFecal® Pro DNA Kit (Qiagen) and the combination of AmpliTest UniProb + AmpliTest RIBO-prep kits (Center for Strategic Planning, Federal Medical-Biological Agency, Russia), both of which outperformed other methods in terms of DNA yield. The QIAamp® Fast DNA Stool Mini Kit (Qiagen) showed minimal losses of low-abundance taxa. These findings can be used for the standardization of gut microbiota DNA extraction methodologies and the development of domestic protocols.