Bacterial contamination occurs in different occupational exposures. Although the level of colony-forming units in airborne samples reveals bacterial exposure, the bacterial origin is not specified. We applied Fourier transform infrared spectroscopy to investigate bacterial species in airborne samples and validated bacterial evaluation in an occupational exposure. As a pilot study, airborn of nurses station (n=40) was assessed according to National Institute of Occupational Safety and Health 0800 method. Bacterial strain in all samples was evaluated by diagnosis gallery tests. Simultaneously, Fourier transform infrared spectroscopy of sampled bacteria was prepared and then classified by principal component analysis. Spectroscopy method was validated for Staphylococcus aureus quantification. For this aim, spectrum band area in different wavenumbers was evaluated for calibration curve plotting and extraction of validation parameters. The second derivatization of spectroscopy data in 1200 to 1500cm-1 presented the best determination for gram-type discrimination by principal component analysis modeling. Moreover 900-1200cm-1 region modeling finely differentiated gram-negative bacteria in airborne samples. Adding spectroscopy area band in 2850 and 2906cm-1 was validated to assess the level of Staphylococcus aureus contamination. Results showed appropriate correlation between the Staphylococcus aureus level in airborne samples analyzed by commercial counting evaluation and Fourier transform infrared spectroscopy method. Fourier transform infrared spectroscopy is a functional technique for assessment of airborne quality in occupational exposure. This analysis was recommended as an alternative method for quantitative and qualitative analysis of bio-aerosols.