Silk fibroin (SF), the primary protein in silkworm silk, has emerged as a promising organic nonlinear optical material due to its unique combination of optical transparency, biocompatibility, and environmental sustainability. In this study, we investigate the nonlinear optical properties of SF thin films using the z-scan technique with femtosecond laser pulses (35 fs, 800 nm, 1 kHz). Our results reveal a strong self-defocusing effect (negative nonlinear refractive index) and significant multiphoton absorption, demonstrating SF’s tunable nonlinear response. Additionally, optical transmittance measurements confirm SF’s partial transparency in the deep UV region, enhancing its potential for second-harmonic generation (SHG) and efficient light frequency conversion. These findings address a key knowledge gap in nonlinear optics, positioning SF as a versatile biopolymer for advanced photonic applications.