The objectives of this study are to investigate, over time, the antimicrobial activity against polymicrobial biofilms and ability to inhibit biofilm formation, of Biodentine (BD) alone and with 5% and 10% sodium diclofenac (DC). The antimicrobial activity of BD alone and modified with 5% and 10% DC against polymicrobial biofilm growth in dentin was determined by a modified direct contact test. The study groups were (1) BD; (2) BD + 5% DC; and (3) BD + 10% DC. The viability of microorganisms after 1 and 4weeks was quantified by means of an ATP assay and flow cytometry. The antibiofilm efficacy of the materials, preventing polymicrobial biofilm formation over time, was assessed by confocal laser scanning microscopy (CLSM). The results obtained with both the ATP test and flow cytometry showed that BD alone and with 5% and 10% DC exerted antibiofilm activity with respect to the control, in the two evaluated times (p < 0.001). Comparison between groups showed a tendency of increased antimicrobial effect, both over time and depending on the DC concentration. These results coincide with those obtained in CLSM analysis, where efficacy increased with time and DC concentration. Biodentine, over time, showed antimicrobial and antibiofilm efficacy on polymicrobial biofilms. The addition of 5% and 10% DC to BD enhanced this effect, in a concentration- and time-dependent manner.