Disinfectants are widely used in healthcare facilities to prevent the occurrence of hospital associated infections (HAIs), especially in intensive care units (ICUs). Microorganisms adhere to different surfaces and become physically structured in biofilms by secreting extracellular polymeric substances (EPSs) which act as an external shield for the microorganisms, reducing the penetration and diffusion of biocidal substances. This study analyzed the presence of phenotypically resistant Gram-negative rods in ICUs and investigated whether biofilms are responsible for the persistence of multidrug-resistant bacteria in these specialist hospital wards. Multidrug-resistant Gram-negative rods were isolated after ICU disinfection and evaluated for susceptibility to three disinfectants [benzalkonium chloride and biguanide (BCB), sodium hypochlorite (NaClO), and hydrogen peroxide (H2O2)] under two different conditions: when they are in a planktonic state (unicellular-life phase) and after induction to form biofilms.