Denture biofilm acts as a potential reservoir for respiratory pathogens, considerably increasing the risk of lung infections, specifically aspiration pneumonia, mainly 48h after hospital admission. The establishment of a straightforward, affordable, and applicable hygiene protocol in a hospital environment for the effective control of denture biofilm can be particularly useful to prevent respiratory infections or reduce the course of established lung disease. To evaluate the anti-biofilm effectiveness of denture cleaning protocols in hospitalized patients. The maxillary complete dentures (MCDs) of 340 hospitalized participants were randomly cleaned once using one of the following 17 protocols (n=20): brushing with distilled water, toothpaste, or neutral liquid soap (controls); immersion in chemical solutions (1% sodium hypochlorite, alkaline peroxide, 0.12% or 2% chlorhexidine digluconate), or microwave irradiation (650 W for 3 min) combined or not with brushing. Before and after the application of the protocols, the biofilm of the intaglio surface of the MCDs was evaluated using two methods: denture biofilm coverage area (%) and microbiological quantitative cultures on blood agar and Sabouraud Dextrose Agar (CFU/mL). Data were subjected to the Wilcoxon and Kruskal-Wallis tests (α=0.05). All 17 protocols significantly reduced the percentage area of denture biofilm and microbial and fungal load (P<0.05). The highest percentage reductions in the area of denture biofilm were observed for 1% hypochlorite solution with or without brushing and for 2% chlorhexidine solution and microwave irradiation only in association with brushing (P<0.05). The greatest reductions in microbial and fungal load were found for the groups that used solutions of 2% chlorhexidine and 1% hypochlorite and microwave irradiation, regardless of the association with brushing (P<0.05). A single immersion for 10 min in 1% sodium hypochlorite, even in the absence of brushing, proved to be a straightforward, rapid, low-cost, and effective protocol for cleaning the dentures of hospitalized patients.