Reflection of electromagnetic waves at multiple interfaces and then absorption in the matrices has been well demonstrated to efficiently improve electromagnetic interference (EMI) shielding of conductive polymer composites (CPC). In this study, a large number of multiple interfaces were constructed in polydimethylsiloxane/multi-walled carbon nanotubes (PDMS/MWCNT) nanocomposites by the incorporation of cotton fibers (CTF). The electromagnetic radiation was efficiently attenuated by the wave reflection at the multiple interfaces and then absorption at the interfaces of PDMS/CTF and CTF/MWCNT in the nanocomposites. The EMI shielding effectiveness (SE) of the PDMS/MWCNT nanocomposites with 2.0 and 3.0 vol% MWCNT increased from ∼16 to ∼30 dB, ∼20 to ∼41 dB by adding 15 vol% CTF, respectively. A relatively dense MWCNT network, which was also formed in the PDMS/MWCNT/CTF nanocomposites because of the volume occupation effect of CTF, resulted in the high electrical conductivity and low percolation threshold. For example, the electrical conductivity of the PDMS/MWCNT nanocomposites with 0.5 vol% MWCNT increased from 1.65 × 10-4 to 0.23 S/m, and the percolation threshold of the composites deceased from 0.44 to 0.2 vol% by the addition of 15 vol% CTF. In addition, the mechanical properties, especially the Young’s modulus and tensile strength, of PDMS/MWCNT nanocomposites were enhanced by the incorporation of CTF and the flexible property was maintained because of the high interfacial interaction between PDMS and CTF and the high aspect ratio of CTF. Furthermore, the samples exhibited highly reliable EMI SE even after bending 6000 times, suggesting the potential application in body protection and flexible electronic devices.