The development of a guided tissue or bone regeneration (GTR/GBR) membrane with excellent performance has been a major challenge in the biomedical field. The present study was designed to prepare a biomimetic electrospun fish collagen/bioactive glass/chitosan (Col/BG/CS) composite nanofiber membrane and determine its structure, mechanical property, antibacterial activity, and biological effects on human periodontal ligament cells (HPDLCs). The effects of this composite membrane on inducing periodontal tissue regeneration were evaluated using a dog class II furcation defect model. It was found that the composite membrane had a biomimetic structure with good hydrophilicity (the contact angle was 12.83 ± 3°) and a tensile strength of 13.1 ± 0.43 Mpa. Compared to the pure fish collagen membrane, the composite membrane showed some degree of antibacterial activity on Streptococcus mutans. The composite membrane not only enhanced the cell viability and osteogenic gene expression of the HPDLCs, but also promoted the expression of RUNX-2 and OPN protein. Further animal experiments confirmed that the composite membrane was able to promote bone regeneration in the furcation defect of dogs. In conclusion, a biomimetic fish Col/BG/CS composite membrane has been developed in the present study, which can induce tissue regeneration with a certain degree antibacterial activity, providing a basis for potential application as a GTR/GBR membrane.