The environmental surveillance of air grilles in clinical areas has not been systematically analysed. Samples were collected from frequently touched items (N= 529), air supply (N= 295) and exhaust (N= 184) grilles in six medical and 11 surgical wards for the cultures of multi-drug-resistant organisms (MDROs): meticillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Acinetobacter baumannii (CRAB) and carbapenemase-producing Enterobacterales (CPE), and isolates were selected for whole-genome sequencing (WGS). The contamination rates were correlated with the colonization pressures of the respective MDROs. From 3rd October to 21st November 2023, 9.8% (99/1008) of the samples tested positive, with MRSA (24.2%, 24/99), CRAB (59.6%, 59/99) and CPE (2.0%, 2/99), being the only detected MDROs. The contamination rate in air exhaust grilles (26.6%, 49/184) was significantly higher than in air supply grilles (5.8%, 17/295; P<0.001). The contamination rate of air exhaust grilles with any MDRO in acute medical wards (73.7%, 14/19) was significantly higher than in surgical wards (12.5%, 4/32; P<0.001). However, there was no difference in the contamination rate of air exhaust grilles between those located inside and outside the cohort cubicles for MDROs (27.1%, 13/48 vs 28.8%, 30/104; P=0.823). Nevertheless, the weekly CRAB colonization pressure showed a significant correlation with the overall environmental contamination rate (r= 0.878; 95% confidence interval (CI): 0.136-0.986; P=0.004), as well as with the contamination rate in air supply grilles (r= 0.960; 95% CI: 0.375-0.999; P<0.001) and air exhaust grilles (r= 0.850; 95% CI: 0.401-0.980; P=0.008). WGS demonstrated clonal relatedness of isolates collected from patients and air exhaust grilles. Air grilles may serve as MDRO reservoirs. Cohort nursing in open cubicles may not completely prevent MDRO transmission through air dispersal, prompting the consideration of future hospital design.