The growing demand for advanced DNA sequencing technologies has stimulated the exploration of novel two-dimensional (2D) materials with exceptional physicochemical properties. In this study, the interactions between DNA nucleobases (adenine, thymine, guanine, and cytosine) and 1H-WSe2 and Janus 1H-WSSe monolayers were investigated using density functional theory with the OpenMX code. The structural, electronic, and optical properties of the nucleobase–substrate systems were systematically analyzed to assess their potential for sequencing applications. Structural relaxation results reveal physisorption behavior, evidenced by minimal bond length changes upon adsorption. Binding energy calculations indicate that guanine exhibits the strongest interaction with 1H-WSe2 (G > T > A > C), whereas thymine shows the strongest binding on Janus 1H-WSSe (T > A > C = G). Charge density difference and Mulliken population analyses confirm weak charge transfer dominated by van der Waals interactions. Density-of-states analysis demonstrates that adsorption does not alter the semiconducting nature of either substrate, preserving their intrinsic electronic characteristics. Optical analyses reveal anisotropic dielectric responses, where pristine 1H-WSe2 exhibits zero-crossing energies at approximately 6.29 eV and 7.19 eV for the out-of-plane (E∥c) polarization and 8.37 eV and 9.03 eV for the in-plane (E⊥c) polarization. Janus 1H-WSSe shows slightly higher values at around 6.74 and 7.66 eV (E∥c) and 8.81 and 9.75 eV (E⊥c). These results highlight the fact that both materials maintain high optical responsiveness with tunable plasmonic excitations upon nucleobase adsorption. Overall, monolayer 1H-WSe2 and Janus 1H-WSSe exhibit stable physisorption and favorable optoelectronic features, making them promising candidates for next-generation DNA sequencing platforms.