| Summary: | Bacteriophage therapy has emerged as a strategy supplementing traditional disinfection protocols to fight biofilms. The aim of the study was to isolate the phages against <i>E. faecalis</i> and to characterize its biological features, morphology, and lytic activity in a formed biofilm model. Methods: <i>E. faecalis</i> ATCC 29212 strain was used for the trial. Two novel vB_Efa29212_2e and vB_Efa29212_3e virulent phages were isolated from urban wastewater and characterized. The <i>E. faecalis</i> biofilm was established in 15 bovine teeth for 21 days. Transmission (TEM) and scanning electron (SEM) microscopes with the colony-forming unit (CFU) counting were used for assessment. Results: Isolated phages differed in morphology. Taxonomy for vB_Efa29212_2e (Siphoviridae, Efquatovirus) and for vB_Efa29212_3e (Herelleviridae, Kochikohdavirus) was confirmed. Both phages were stable at a temperature range of 4–50 °C and showed a different tolerance to chemicals: 15% EDTA, 1–3% sodium hypochlorite, and chlorhexidine. SEM analysis showed distortion of bacteria cells after phage inoculation, which proved the lytic activity against <i>E. faecalis</i>. A 54.6% reduction in the <i>E. faecalis</i> biofilm confirmed bacteriophage efficacy against isolates in the ex vivo model. Conclusions: Results strongly support the concept that phage therapy has a real therapeutic potential for the prevention and treatment of <i>E. faecalis</i>-associated infections.
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