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Background During the Hajj season, respiratory symptoms are very common among pilgrims. Here, we investigated the viable bacterial population in air samples collected around the slaughterhouses used during the Hajj. Methods and results We collected air samples on three days from four different sites: slaughterhouses at Al-Kakia, Al-Meaisim and Al-Sharaia, and from a waste disposal area designated for the remnants of slaughter. Samples were cultured on blood agar plates for 48 h, and bacterial isolates were identified using MALDI-TOF MS. A dendrogram using the spectra of the unidentified bacterial species was constructed, and PCR amplification and sequencing of the 16S rRNA gene was performed for one isolate per cluster. In total, 2500 colonies appeared on the nutrient agar plates, and 244 were purified for further analysis. Good identification was obtained for 202 (83%) isolates by MALDI-TOF MS. The most common genera were Bacillus (n = 94, 45%) and Staphyloccocus (n = 55, 26%). Poor identification was obtained for 42 (17%) isolates, and their spectra clustering revealed that these isolates belonged to 10 species. Four of these were considered to be new species. Conclusions During the Hajj, the air was contaminated by many environmental bacterial agents, and MALDI-TOF MS was successfully adapted for their rapid identification. [ABSTRACT FROM AUTHOR]