The purpose of this study was to evaluate the bactericidal effect of 270 nm UV-C light-emitting diode (LED) light delivered through a newly designed prototype device with thin optical fiber against Enterococcus faecalis (E. faecalis). The prototype device, developed to integrate UV-C light into a thin optic fiber (diameter 124 µm) connected to a UV-C LED (Luminous Device; Sunnyvale, CA, USA) via a specialized double-lens system that focuses divergent light to achieve a 65 mm working distance and a numerical aperture of 0.22. E. faecalis, was cultured at 37 °C under aerobic conditions for 24 h. The UV-C LED optical fiber was positioned 10 mm above the bacterial culture prepared in the wells of a 96-well plate. The E. faecalis cells were exposed to UV-C irradiation for 0, 10, 30, 60, 90, 120 and 180 s. Following irradiation, the OD600 values were measured after incubation at 37 °C for an additional 24 h. The data were statistically analyzed using one-way ANOVA, followed by Tukey’s honestly significant difference (HSD) test at a significance level of 0.05. UV irradiation at 270 nm significantly reduced E. faecalis growth in a time-dependent manner (p < 0.05). No significant changes were observed at 0 and 10 s, while peak reductions occurred at 120 and 180 s, with effects beginning at 30 s and increasing over time. The 270 nm UV-C wavelength was highly effective in bactericidal action against E. faecalis. The custom-designed UV-C delivery system effectively integrated the light source into a thin optical fiber, allowing for efficient UV-C light transmission and demonstrating its potential for application in narrow spaces such as root canals.