The aim of this study was to investigate in vitro the antimicrobial effect and diffusion against E. faecalis of new intracanal medications on the external root surface. The medications tested were a placebo gel (PC); the new formulations with either 3% nitrofurantoin (NIT) or 3% doxycycline hydrochloride (DX) and 2% chlorhexidine (CHX) gel as positive control. The new formulations were tested using the traditional agar diffusion test (ADT) and an adapted agar diffusion method (AADM), where the teeth were filled with the medications and left to diffuse on agar surface seeded with E. faecalis. In the ADT, the larger zones of microbial growth inhibition were seen in DX, followed by CHX and NIT. In the AADM test only DX and CHX showed antimicrobial effect. Statistically significant differences between groups were observed by the Kruskal-Wallis test (2=47.126; p<0.001). The new intracanal formulations with DX and NIT have demonstrated antimicrobial effect against E. faecalis, but only DX was able to diffuse through the dentinal tubules and exert antimicrobial effect outside the roots.