Background: This in vitro study compared the effectiveness of polytetrafluoroethylene (PTFE) tape and a conventional cotton retraction cord for gingival displacement and evaluated the mechanical and microstructural characteristics of PTFE tape. Methods: Eight standardized prepared teeth with artificial gingiva were evaluated under three conditions: control, cotton retraction cord, and PTFE tape. After each condition, impressions were digitized using a CAD/CAM system. Sulcular width was measured at four predefined locations and averaged for statistical analysis. PTFE tape was also evaluated by tensile testing before and after sterilization and by scanning electron microscopy (SEM). Results: Mean sulcular widths were 0.716 ± 0.118 mm (control), 0.851 ± 0.140 mm (cotton cord), and 0.907 ± 0.099 mm (PTFE). Repeated-measures ANOVA showed a significant effect of the displacement method (p < 0.001), with PTFE producing significantly greater sulcular enlargement than cotton cord. Qualitative SEM analysis revealed a more homogeneous impression surface morphology following PTFE removal. Sterilization did not significantly affect the tensile properties or microstructural integrity of PTFE tape. Conclusions: Under standardized in vitro conditions, PTFE tape produced the highest gingival sulcular enlargement and maintained favorable mechanical and microstructural properties, supporting its potential as an alternative gingival displacement material. Further clinical studies are required to confirm these findings.