While periodontal ligament cells are sensitive to their 3D biomechanical environment, only a few 3D in vitro models have been used to investigate the periodontal cells mechanobiological behavior. The objective of the current study was to assess the capability of a 3D fibrous scaffold to transmit a mechanical loading to the periodontal ligament cells. Three-dimensional fibrous polycaprolactone (PCL) scaffolds were synthetized through electrospinning. Scaffolds seeded with human periodontal cells (103 mL−1) were subjected to static (n = 9) or to a sinusoidal axial compressive loading in an in-house bioreactor (n = 9). At the end of the culture, the dynamic loading seemed to have an influence on the cells’ morphology, with a lower number of visible cells on the scaffolds surface and a lower expression of actin filament. Furthermore, the dynamic loading presented a tendency to decrease the Alkaline Phosphatase activity and the production of Interleukin-6 while these two biomolecular markers were increased after 21 days of static culture. Together, these results showed that load transmission is occurring in the 3D electrospun PCL fibrous scaffolds, suggesting that it can be used to better understand the periodontal ligament cells mechanobiology. The current study shows a relevant way to investigate periodontal mechanobiology using 3D fibrous scaffolds.