Periodontitis is an inflammatory infection caused by bacterial plaque accumulation that affects the periodontal tissues supporting the teeth. Current treatments lack bioactive signals to induce tissue repair and coordinated regeneration of all the periodontal tissues; thus, alternative strategies are needed to improve clinical outcomes. Cell-derived extracellular matrix (ECM) has been used in combination with biomaterials to enhance their biofunctionality for tissue engineering (TE) applications. In this work, bioactive cell-derived ECM-loaded electrospun polycaprolactone/chitosan (PCL-CTS) nanofibrous scaffolds were developed using lyophilized decellularized ECM (dECM) derived from human Periodontal Ligament Stem Cells (PDLSCs). This work’s aims were to fabricate and characterize cell-derived ECM electrospun PCL-CTS scaffolds and to evaluate their capacity to enhance the proliferation of seeded PDLSCs, envisaging future periodontal TE applications.