The present study aimed to investigate whether inhalation of inactivated‑Mycobacterium phlei could prevent airway hyperresponsiveness and airway eosinophilia. A total of 24 male Balb/c mice were randomly divided into three groups: Normal control group (group A), asthma model group (group B) and the intervention group (group C), (8 mice/group). Group A mice were sensitized and with challenged saline and group B with ovalbumin (OVA). Group C mice were administered with aerosol Mycobacterium phlei once daily prior to the allergen challenge. Airway responsiveness in each group was assessed. All the animals were sacrificed and lung tissues, blood samples and bronchoalveolar lavage fluid (BALF) were harvested. Cell fractionation and differential cells were counted in serum and BALF. HE staining and alcian blue/periodic acid Schiff staining were used to measure airway eosinophilic inflammation and mucus production. The levels of the cytokines IL‑5, IL‑13 and IgE were measured in lung and BALF as determined by ELISA and reverse transcription‑quantitative polymerase chain reaction assays. The results indicated that inactivated‑Mycobacterium phlei suppressed the airway hyperresponsiveness and mitigated airway eosinophilia induced by a methacholine challenge, and significantly reduced the levels of cytokines IL‑5 and IL‑13 in lung tissue and IgE level in BALF when compared with the OVA‑sensitized mice. In conclusion, inhalation of inactivated‑Mycobacterium phlei could reduce OVA‑induced airway hyperresponsiveness and may be a potential alternative therapy for allergic airway diseases.