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The ability to control the assembly of nanoparticles on substrates used in plasmon-enhanced spectroscopy continues to drive research in the field of nanofabrication. Here we describe the use of fungi as soft biotemplates to fabricate nanostructured microtubules with gold and gold-silver nanoparticles with potential applications as sensors and biosensors. In the first step, spores of the filamentous fungus Cladosporium sphaerospermum were inoculated in a suspension of gold nanoparticles, forming stable microtubules of gold nanoparticles during fungus growth. These materials were exposed to a second suspension of silver nanoparticles, resulting in complexes multilayers structures of gold and silver nanoparticles, which were evaluated as substrates for surface-enhanced Raman scattering (SERS) using small amounts of thiophenol as probe molecules directly on the microtubules. Both gold and the gold-silver substrates provide the SERS effect.