Aim To evaluate the biocompatibility, induction of mineralization and antimicrobial activity of experimental intracanal pastes based on two glass and glass‐ceramic materials. Calcium hydroxide (Ca(OH)2) paste was used as the positive control. Methodology The glass‐ceramic powder two‐phased Biosilicate (BS‐2P) and F18 bioactive glass were mixed with distilled water (ratio 2 : 1), inserted in polyethylene tubes and implanted in the subcutaneous tissues of 16 rats. Empty tubes were used as negative control. After 7 and 30 days (n = 8), the rats were euthanized for haematoxylin–eosin, von Kossa, polarized light and osteopontin (OPN) immunolabeling analysis. Direct contact tests using a suspension of each paste were performed with Enterococcus faecalis planktonic cells to evaluate antimicrobial activity (24 h of contact), in a pilot study. The number of CFU mL−1 was calculated for each group. The antimicrobial analysis data were submitted to one‐way anova and Tukey tests, whilst biocompatibility and immunohistochemical data were submitted to the Kruskal–Wallis and Dunn tests (P < 0.05). Results Most specimens of the control, BS‐2P and Ca(OH)2 groups were associated with moderate inflammation seven days following implantation, whilst F18 was associated with moderate to severe inflammation, without differences amongst the groups (P > 0.05). At 30 days, most specimens of control, F18 and BS‐2P groups had mild inflammation, whilst Ca(OH)2 had mild to moderate inflammation; however, no differences were determined amongst the groups (P > 0.05). The fibrous capsule was thick at 7 days, becoming thin at 30 days. All pastes induced von Kossa‐positive structures and were birefringent to polarized light. At seven days, the BS‐2P group had significantly more OPN immunolabeling compared to the control and Ca(OH)2 groups (P < 0.05). At 30 days, the F18 group had significantly more OPN immunolabeling compared to the control and Ca(OH)2 groups (P < 0.05). All pastes reduced the total number of E. faecalis; however, the reduction was only significant when comparing BS‐2P and Ca(OH)2 groups to the control (P < 0.05). Only calcium hydroxide eliminated E. faecalis. Conclusions Experimental BS‐2P and F18 pastes were biocompatible, stimulated biomineralization and induced significant OPN immunolabeling compared to Ca(OH)2. Only the BS‐2P paste demonstrated antimicrobial activity comparable to Ca(OH)2.