The discovery that the lung harbors a diverse microbiome, as revealed by next‑generation sequencing, has significantly altered our understanding of respiratory health and disease. Despite the association between the lung microbiota and disease, the nature of their relationship remains poorly understood, and culture isolation of these microorganisms could help to determine their role in lung physiology. Current procedures for processing samples from the lower respiratory tract have been shown to affect the viability of microorganisms, so it is crucial to develop new methods to improve their survival. This study aimed to improve the isolation and characterization of lung microorganisms using a bead‑beating homogenization method in a mouse model. Microsphere diameter and bead‑beating time affected the survival of the microorganisms (E. coli, S. aureus and C. albicans). Using 2.3 mm diameter microspheres for 60 s of bead‑beating promoted the survival of both bacteria and yeast strains. After intratracheal instillation of these microorganisms in mice, approximately 70% of the cells were recovered after the tissue homogenization. To assess the efficiency of the proposed method, the diversity of bacteria was compared between the homogenate and lung tissue samples. Ninety‑one genera were detected in the lung tissue, and 63 in the homogenate. Bacterial genera detected in the homogenate represented 84% of the total abundance of the microbiota identified in the lung tissue. Taken together, these results demonstrate that the tissue homogenization process developed in this study recovered the majority of the microorganisms present in the lung. This study presents a bead‑beating homogenization method for effective cultivation of lung tissue microorganisms, which may help to improve the understanding of host–microbe interactions in the lung.