Background: Healthcare-associated infections (HAIs) represent one of the main causes for the morbidity and mortality in the intensive care unit (ICU). In this study, we aimed to verify the presence, type, and antimicrobial susceptibility of bacteria in ICU settings, before and after disinfection procedures, and analyze the risk of contamination related to these bacteria in each area of the ICU.Methods: The study was conducted in the ICU of a medium-sized hospital in Brazil from February 2019 to February 2020. Samples were obtained from the surfaces of beds, bathrooms, pantries, sinks, pharmacies, administrative areas, and floors, before and after the disinfection process, with 1% benzalkonium chloride and biguanide mixture (BCB). The samples were processed and inoculated in different culture media for the selective isolation of strains of clinical interest. In addition, phenotypic identification and antimicrobial susceptibility tests were performed using the VITEK® 2 system. We grouped different environmental, temporal, and microbial factors and characteristics to calculate the risk of contamination before disinfection (Rbd) and after disinfection (Rad), as well as the total risk (Rtt) and real risk observed (Foldrisk) in the ICU.Results: Gram-positive cocci and rods, gram-negative rods of the Enterobacteriaceae family, and non-fermenting gram-negative rods were recovered after disinfection and were found to be widely distributed. Higher bacterial diversity and frequency of resistance were observed, mainly the resistance of gram-positive bacteria to cephalosporin (p < 0.05) and lincosamides (p < 0.0001), and gram-negative bacteria to quinolones (p < 0.0001) and aminoglycosides (p < 0.05). The pantry, pharmacy, and beds showed major risks of contamination after disinfection (Rad), ranging from intermediate to very high-risk levels. Foldrisk for the pantry and beds presented a significant increase in two and three risk levels, respectively, after the disinfection process.Conclusions: Our results demonstrate the presence of bacterial pathogens with multidrug-resistant profiles after disinfection with a higher risk of contamination, indicating that challenges still exist in the deployed disinfection protocols in the ICU settings, which are associated with the increased critical risk of HAIs after the disinfection procedure.