The discovered light modulation capabilities of diatom silicious valves make them an excellent toolkit for photonic devices and applications. In this work, a reproducible surface-enhanced Raman scattering (SERS) enhancement was achieved with hybrid substrates employing diatom silica valves coated with an ultrathin uniform gold film. Three structurally different hybrid substrates, based on the valves of three dissimilar diatom species, have been compared to elucidate the structural contribution to SERS enhancement. The comparative analysis of obtained results showed that substrates containing cylindricalAulacoseirasp. valves achieved the highest enhancement, up to 14-fold. Numerical analysis based on the frequency domain finite element method was carried out to supplement the experimental results. Our results demonstrate that diatom valves of different shapes can enhance the SERS signal, offering a toolbox for SERS-based sensors, where the magnitude of the enhancement depends on valve geometry and ultrastructure.