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Bacterial biofilms, formed on biotic or abiotic surfaces, can lead to serious environmental or medical problems. Therefore, it is necessary to find novel antimicrobial agents to combat biofilms, or more effective combinations of existing biocides. In this study, initial biofilms of Pseudomonas aeruginosa ATCC 9027 and Staphylococcus aureus ATCC 6538 in the presence of xylitol or xylitol and isothiazolones were determined using crystal violet staining in 96-well microplates and confocal laser scanning microscopy. Xylitol and isothiazolones exhibited enhanced synergistic inhibition of initial biofilm formation, and also the structure and production of extracellular polymeric substances by P. aeruginosa ATCC 9027 and S. aureus ATCC 6538 in a dose-dependent manner. In addition, xylitol and isothiazolones inhibited and restored the swimming motility of P. aeruginosa ATCC 9027, respectively. These findings show that a combination of xylitol and isothiazolones exerts pronounced antimicrobial activity against P. aeruginosa and S. aureus biofilms and may be applicable for preventing or reducing bacterial biofilms in vitro.