The inactivating efficiency using membrane-less electrolyzed water (MLEW) spraying was evaluated against two airborne strains, Staphylococcus aureus and λ virus aerosols, in an indoor environment-simulated chamber. The air exchanged rate (ACH) of the chamber was controlled at 0.5 and 1.0 h–1. MLEW with a free available chlorine (FAC) concentrations of 50 and 100 ppm were pumped and sprayed into the chamber to treat microbial pre-contaminated air. Bioaerosols were collected and cultured from air before and after MLEW treatment. The first-order constant inactivation efficiency of the initial counts of 3 × 104 colony-forming units (CFU or PFU)/m3 for both microbial strains were observed. A higher FAC concentration of MLEW spraying resulted in greater inactivation efficiency. The inactivation coefficient under ACH 1.0 h–1 was 0.481 and 0.554 (min–1) for Staphylococcus aureus of FAC 50 and 100 ppm spraying. In addition, increasing the air exchange rate also improved the inactivation rate. The inactivation coefficient of FAC 100 ppm spraying for Staphylococcus aureus was 0.412 and 0.403 (min–1) under ACH 1.0 and 0.5 h–1. These results indicated that MLEW spraying is likely to be effective in minimizing microbial airborne contamination, especially for poorly ventilated spaces.