Objectives This study aimed to evaluate the antibiofilm effect of calcium hydroxide (CH), 0.5% iodine potassium iodide (IKI), and 0.5% cetrimide (CTR), alone and in combinations on one-week and three-week-old biofilms. Materials and methods Gingival plaque was collected, and biofilms were grown anaerobically. Biofilms were exposed to each of the three medicaments and their combinations for one day, one week, and two weeks. Proportions of dead and live bacteria in the biofilms were evaluated. Results The killing of bacteria by different medicaments in the three-week-old biofilm was lower than in the one-week-old biofilm (<0.05). The efficacy of IKI and CTR in killing bacteria was weaker than that of CH, but the highest efficacy in killing was achieved when all three were combined (<0.05). There was no significant difference in the antibiofilm effect between a day's exposure to the mixture of the three medicaments and one or two weeks of treatment with CH alone (>0.05). Conclusions Three-week-old biofilms are more resistant to medicaments than one-week-old biofilms. Combining IKI and CTR with CH resulted in a stronger antibiofilm effect than using CH alone. Mixing the three medicaments may enable obtaining the desired clinical effect in a shorter exposure time.