This study was conducted at one of the largest poultry companies in Kuwait during November and December 2019 to evaluate the microbiological threats ofEscherichia coli(APEC),Salmonellaspp., andAspergillus fumigatusto chickens in fattening houses by counting and identifying the microorganisms by culturing and pyrosequencing analysis. During the fattening cycle, the temperature and humidity ranged between 23.6°C and 29°C and 64.1% and 87.1%, respectively. The total bacterial population andAspergillus fumigatusmeasured in the indoor and outdoor air exhibited a linear relationship during the fattening cycle. The total bacterial andAspergillusconcentrations determined during the cycle ranged between 150 and 2000 CFU/m3and 0 and 1000 CFU/m3, respectively.E. coliandSalmonellaspp. concentrations determined during the cycle ranged between 1 and 220 CFU/m3and 4 and 110 CFU/m3, respectively. Pyrosequencing analysis of the air inside the houses at the end of the cycle revealed extensive biodiversity in the microorganisms, detecting 32 bacterial genera and 14 species. The identified species belonging to the generaCorynebacterium,Haemophilus,Streptococcus,Veillonella, andAspergilluswere identified as potentially affecting human and broiler health. The emission of potentially pathogenic bacteria to the outdoor environment from chicken housing can pose a considerable risk to human health and environmental microbial pollution. This study could guide the development of integrated control devices for monitoring microbes in broiler production facilities during chicken collection for transport to slaughterhouses.