Healthcare-associated infections (HAIs) represent a global challenge and an even more staggering concern when related to microorganisms capable of resisting and surviving for long periods in the environment, such as Acinetobacter spp. Strategies that allow a reduction of pathogens from hospital environments represent an additional barrier in infection control protocols, minimizing transmission to hospitalized patients. Considering the antimicrobial properties of copper, here, the bacterial load and the presence of Acinetobacter spp. were monitored on high handling surfaces covered by 99.9% copper films on intensive and non-intensive care unit bedrooms in a tertiary care hospital. Firstly, copper-coated films were able to inhibit the adhesion and biofilm formation of A. baumannii strains in in vitro assays. On the other hand, Acinetobacter spp. were isolated from both copper-coated and uncoated surfaces in the hospital, although the majority was detected on surfaces without copper. All carbapenem-resistant A. baumannii isolates identified harbored the blaoxa-23 gene, while the A. nosocomialis isolates were susceptible to most antimicrobials tested. All isolates were susceptible to polymyxin B. Regarding the total aerobic bacteria, surfaces with copper-coated films presented lower total loads than those detected for controls. Copper coating films may be a workable strategy to mitigate HAIs, given their potential in reducing bacterial loads in nosocomial environments, including threatening pathogens like A. baumannii.