Denture plaque is a biofilm composed of various microorganisms aggregated with saliva. Various denture cleansers and cleaning apparatuses have been developed and studied. However, the optimum water temperature for denture cleaning is unknown. Therefore, the present study investigated the effects of water temperature during ultrasonic denture cleaning. In vitro, resin disks with artificial Candida albicans biofilm were pressed onto Candida GE media after ultrasonic cleaning with water at different temperatures for 5 min. The media were subsequently cultured at 37°C for 24 h. The colonies formed were observed and colony areas were quantified using ImageJ software (US National Institutes of Health, Bethesda, MD, USA). In situ, the bacterial count and degree of cleanliness on the tissue surface of maxillary dentures were measured before and after ultrasonic cleaning with water at different temperatures for 5 min. Changes in bacterial counts and cleanliness were calculated for each temperature. The ratio of the area occupied by bacterial colonies in vitro and reduction rates in situ after cleaning with warm water were markedly less than those observed after cleaning with cold water. Therefore, ultrasonic denture cleaning with warm water is more effective.