Aim: The current study investigated the performance of 4 widely used DNA extraction kits using different types of high (stool) and low biomass samples (chyme, broncho alveolar lavage and sputum). Methods: Qiagen Powerfecal Pro DNA kit, Macherey Nucleospin Soil kit, Macherey Nucleospin Tissue Kit and MagnaPure LC DNA isolation kit III were evaluated in terms of DNA quantity, quality, diversity and composition profiles. Results: The quantity and quality of DNA varied among the four kits. The microbiota of the stool samples showed similar diversity and compositional profiles for the 4 kits. Conclusion: Despite differences in DNA quality and quantity, the 4 kits yielded similar results for stool samples, while all kits were not sensitive enough for low biomass samples.