Objective: This study aimed to investigate the possible topical effect of a broad-spectrum antibiotic on dental biofilm formed in situ in the absence or presence of sucrose. Methods: A crossover study was conducted in three phases of 14 days each, during which 11 volunteers wore palatal devices containing 6 enamel blocks covered with meshes to allow biofilm formation. Dental blocks were extraorally submitted to a 20% sucrose solution at three different frequencies of exposure (0, 3 and 8 times/day), and to a suspension of amoxicillin/clavulanate potassium (A/CP) or a placebo (P) suspension at an 8-hour time interval application regimen. On the 14th day of each phase, biofilms were collected for microbiological (conventional culture) and molecular (Denaturing Gradient Gel Electrophoresis - DGGE) analyses. Results: In the absence of sucrose exposure (SE) and at the 3-time daily frequency, dental biofilms treated with A/CP showed lower total biofilm weight and lower counts of total microbiota than the ones treated with P (p>0.05). A/CP presented higher counts of Candida spp. when compared with P in the presence of SE, especially at the 8-time daily frequency (p0.05), regardless of SE. However, DGGE profiles demonstrated large interindividual variability. Conclusion: Both conventional culture and DGGE have demonstrated some differences on total microbiota of dental biofilms when exposed to the A/CP or P suspensions, mainly in the absence of sucrose, which suggests a possible topical effect of the sugar-free A/CP suspension on dental biofilm. © 2013 Pierro et al.