The incorporation of titanium dioxide (TiO₂) nanoparticles into polyethylene terephthalate (PET) fibers represents a promising strategy for developing multifunctional materials combining enhanced optical properties with antimicrobial activity. This study employed variable wavelength automated interferometry (VAWI) with the Fourier transform method (FTM) across the visible spectrum (400-700 nm) to comprehensively characterize PET fibers functionalized with 2.0 wt% TiO₂ nanoparticles. The nanocomposite fibers demonstrated substantial property enhancements compared to pristine PET: 18.5% birefringence increase, 15.3% enhancement in parallel dispersion, 18.4% improvement in molecular orientation, and 68% increase in stress-optical coefficient. Cauchy dispersion analysis revealed enhanced wavelength-dependent optical response with dispersion anisotropy ratio increasing from 1.63 to 1.82. Antimicrobial efficacy testing via shaking flask methodology demonstrated robust broad-spectrum activity with 75.8-85.4% growth reduction against diverse pathogens, attributed to photocatalytic reactive oxygen species generation. These findings establish quantitative structure-property-performance relationships essential for rational design of multifunctional fiber materials with tailored optical, mechanical, and biological properties for advanced textiles, photonic devices, and biomedical applications.