Different steps and conditions for DNA extraction for microbiota analysis in sputum have been reported in the literature. We aimed at testing both dithiothreitol (DTT) and enzymatic treatments of sputum samples and identifying the most suitable DNA extraction technique for the microbiota analysis of sputum. Sputum treatments with and without DTT were compared in terms of their median levels and the coefficient of variation between replicates of both DNA extraction yield and real-time PCR for the 16S rRNA gene. Treatments with and without lysozyme and lysostaphin were compared in terms of their median levels of real-time PCR for S. aureus. Two enzyme-based and three beads-based techniques for DNA extraction were compared in terms of their DNA extraction yield, real-time PCR for the 16S rRNA gene and microbiota analysis. DTT treatment decreased the coefficient of variation between replicates of both DNA extraction yield and real-time PCR. Lysostaphin (either 0.18 or 0.36 mg/mL) and lysozyme treatments increased S. aureus detection. One enzyme-based kit offered the highest DNA yield and 16S rRNA gene real-time PCR with no significant differences in terms of alpha-diversity indexes. A condition using both DTT and lysostaphin/lysozyme treatments along with an enzymatic kit seems to be preferred for the microbiotaanalysis of sputum samples.