Gram-negative bacteria (GNB) including Acinetobacter baumannii, Pseudomonas aeruginosa and Legionella have emerged as causative agents of many problematic infections in healthcare settings worldwide. This study was designed to investigate the presence of GNB in hospital air as a potential source for spread and transmission of these bacteria, by a rapid detection method. A total of 51 air samples were taken from different wards of four hospitals over a period of eight months. Air samples were collected using an all-glass impinger, and analyzed for the presence of GNB. Detection of P. aeruginosa and Legionella spp. was performed with a nested PCR assay using specific primer sets of the 16S rRNA gene region of the bacteria. For detection of A. baumannii, PCR assay with the specific primers of the inherent blaOXA-51 gene was performed. A. baumannii was the most frequently (29/51) detected gram negative microorganism in hospital air followed by P. aeruginosa (15/51). The lowest detection frequency was related to Legionella (9/51), which was not found in air samples of surgery wards. Intensive care units and operating teaters were the high-risk areas due to the more presence of GNB in these wards. The results of this study revealed the presence of GNB in various hospital wards. The results highlight the usefulness of PCR-monitoring of hospital air as a rapid and reliable tool for identification of GNB, to prevent and control the nosocomial infections especially for protection of vulnerable patients.