In the present research, for the first time, lycopodium as a novel nanofiller was incorporated into a polyvinylidene fluoride matrix to fabricate lycopodium/polyvinylidene fluoride flat-sheet membrane for desalination applications by vacuum membrane distillation process. The prepared lycopodium/polyvinylidene fluoride membranes and lycopodium were characterized by field emission scanning electron microscopy, X-ray diffraction, Fourier transform infrared, energy dispersive X-ray, and mapping analyses. Water contact angle and liquid entry pressure measurements were also performed. Response surface methodology was applied to optimize membrane structure and performance. The optimized lycopodium/polyvinylidene fluoride membrane exhibits superior performance compared to the neat polyvinylidene fluoride membrane in terms of flux, salt rejection, water contact angle, and hydrophobicity. In vacuum membrane distillation experiments, using a 15000 ppm NaCl solution as a feed at 70 °C, the neat polyvinylidene fluoride membrane, optimum membrane, and agglomerated membrane (with high lycopodium loading) demonstrated 3.80, 25.20, and 14.83 LMH flux and 63.30%, 99.99%, 99.96% salt rejection, respectively. This improvement in flux and salt rejection of the optimized membrane was related to the presence of lycopodium with hydrophobic nature and interconnected nano-channels in membrane structure. It was found that lycopodium, as the most hydrophobic material, effectively influences the membrane performance and structure for membrane distillation applications.