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The purpose is to evaluate the antibacterial effects of the silver nanoparticles (AgNPs) (Nanografi, METU Teknokent, Ankara, Turkey) mixed with calcium hydroxide (Ca(OH)2) (Ultracal XS, Ultradent, St Louis, US) or chlorhexidine gel (CHX) (Gluco-Chex, Cerkamed, Stalowa Wola, Poland) against a multispecies biofilm, by confocal laser scanning microscopy (CLSM) and culture-based analysis. Dentine blocks were inoculated with Enterococcus faecalis, Streptococcus mutans, Lactobacillus acidophilus and Actinomyces naeslundii for 1 week. Infected dentine blocks were randomly divided into groups according to medication; saline solution (SS), Ca(OH)2, Ca(OH)2 + AgNP, 2%CHX gel and 2%CHX gel + AgNP and time of application: 1 and 7 days (all groups, n = 5). Bacterial samples were collected before and after medication to quantify the bacterial load. Biofilm elimination was quantitatively analyzed by Live/Dead BacLight Bacterial Viability staining and CLSM. The addition of AgNPs to Ca(OH)2 increased the effectiveness of medicament in terms of bacterial reduction in both application times (1 and 7 days) (p < 0.05: ANOVA, Tukey's test) according to culture-based analysis. The CLSM images revealed that mixture of AgNP with CHX killed significantly more bacteria when compared with all other medicaments at 1- and 7-day application times (p < 0.05 and p > 0.05, respectively: Kruskal–Wallis, Dunn post hoc tests). The efficacy of Ca(OH)2 mixed with AgNPs was superior to Ca(OH)2 used alone in both application times (p < 0.05) according to CLSM analysis. The present study put forth the potential use of AgNPs mixed with Ca(OH)2 or CHX on multispecies (Enterococcus faecalis, Streptococcus mutans, Lactobacillus acidophilus and Actinomyces naeslundii) biofilm in 1 and 7day application periods. [ABSTRACT FROM AUTHOR]