Introduction Intracanal medications must have antimicrobial properties, in addition to reaching the natural complexities of the periapical tissues, to help in their recovery. Considering the need for more studies about associations among these medications, we aimed to analyze the antimicrobial activity of daptomycin, alone and associated with Triple Antibiotic Paste (TAP), through the penetrability of bovine dentin against four Enterococcus faecalis samples: one standard American Type Culture Collection (ATCC) 29212 sample and three clinical samples. Methods Eighty-three bovine freshly extracted teeth were standardized at 15 mm and prepared to file 45, and then sterilized. The roots received, randomly, one of five treatments (n=16/group). Group 1: TAP; Group 2: daptomycin; Group 3: TAP+daptomycin; Group 4: calcium hydroxide; and Group 5: propylene glycol. The treated roots were inserted into a previously prepared orifice in the base of Petri dishes, which were, twice a week, filled with solid medium inoculated with E. faecalis (five fresh inoculated mediums/15 days). After 24 h incubation with each new inoculum, a reading was performed to determine the presence/absence of inhibition zones around the roots. A total of five readings, on days 1, 4, 8, 11, and 15, were performed for each plate. Three untreated roots with culture medium without inoculum represented the sterility control group. Results Group 3 (TAP associated with daptomycin) was the group responsible for the highest number of inhibition zones and the only one capable of inhibiting all E. faecalis strains in at least one of the readings. Comparing TAP (group 1) with daptomycin (group 2) alone, group 1 presented a higher number of inhibition zones than group 2. Groups 4 and 5 (calcium hydroxide and propylene glycol) did not present inhibition halos for any of the evaluated E. faecalis strains. Absence of microbial growth was observed for all roots in the sterility control group. Conclusions This study showed, through the penetrability of bovine dentin, that the association between TAP and daptomycin improved the antimicrobial effectiveness of TAP, demonstrating a possible synergistic activity against E. faecalis (ATCC and clinical samples). TAP associated with daptomycin can be considered as a potential intracanal medication, but only as a last resort.