Spider silk has biocompatibility and biodegradability properties and is known for the mechanical, physical and chemical properties that make it a promising building block in the development of novel biofibers. Its unique properties partially result from the repetitive polypeptide sequences that compose the silk proteins. The strength is related to the polyalanine motifs organized into β-sheet structures, and the elasticity is attributed to glycine-rich regions, β turns and 310 helix structures. Some alcohols were shown to induce β-sheet formation in spidroins and spider silk films, while water increases the overall structure ordering of silkworm fibroins. Furthermore, fiber stretching induces β-sheet formation in synthetic spider fibers. However, there is a lack of information relating the physical and mechanical behaviors that might contribute to improving the microstructure and performance of synthetic fibers. In this work, we reported the surface nanostructure and the nanomechanical behavior of synthetic spider fibers, which were composed of modified recombinant proteins to combine strength and extensibility motifs. Our present study evaluated synthetic fibers qualitatively and quantitatively, and indicated that atomic force microscopy (AFM) and scanning electron microscopy (SEM) were complementary tools to describe particular details of the surface structure and the mechanical features of synthetic spider fibers. Therefore, AFM and SEM would support the development of spinning systems and the characterization of novel biomaterials. Synthetic spider fibers have particular structural and mechanical features that can be improved through extrusion treatments. The surface irregularity, elasticity and strength of fibers are likely to be dependent on their molecular organization and an alcohol bath appears to initiate the transition of random coil structures to β-sheets, resulting in smoother, stronger and more elastic fibers. Atomic force microscopy/force spectroscopy complemented by scanning electron microscopy was able to qualitatively describe synthetic fibers and quantify their roughness and mechanical properties, representing a significant step toward spider silk characterization, and for its application as a biomaterial.