Purpose: It is a challenge to find a better microorganisms DNA extraction method for samples taken from the lower airways for metagenomic sequencing, as the concentrations of bacteria in the alveoli and small airways are likely considerably less than that of the mouth or lower digestive tract. Background DNA from the host, and extraction biases can significantly interfere with microbiota assessment and increase the cost of sequencing. This study aimed to develop an optimized DNA extraction method, which would enable a higher concentration of microbial DNA to be extracted from the samples. Methods: We compared the microbiota profiles of the lower airway communities in twelve individuals with IIP. DNA was extracted using three different extraction methods: QIAamp UCP PurePathogen Blood Kit named kit3 in this study, QIAamp UCP Pathogen Mini Kit named kit2, and QIAamp DNA Microbiome Kit named kit1. DNA libraries were constructed according to the manufacturer's instructions (Illumina). The same workflows from Illumina were used to perform cluster generation, template hybridization, isothermal amplification, linearization, blocking, denaturing, and hybridization of the sequencing primers. Raw data was uploaded to MG-RAST v3 and analyzed. Results: A great number of bacterium inhabits the lower airways of patients with IIP, though there is no airway infection. More bacterium was found in mouth or upper airway. DNA concentrations of DNA samples isolated with kit1 with Benzonase were significantly lower than those isolated with the other two kits for BALF and mouthwash samples. Moreover, the ratio of human genome in clean reads of samples isolated with kit1 with Benzonase was remarkably smaller than those isolated with kit2 and kit3. The relative abundance of total bacteria, the total number of taxa, and the relative abundance of taxa in BALF samples as opposed to mouthwash samples with kit1 were significantly higher than for those extracted the other kits. Conclusion: A microbial DNA extraction method with pretreatment of depletion of host nucleic acid by Benzonase can enable a higher yield of microbial DNA from samples with a higher fraction of host cells to be obtained. The lower airways of patients with IIP without airway infection were inhabited by a great number of bacterium.