For its powerful antimicrobial properties, nanosilver is the most widely used nanoparticle in commercial odor-neutralizing and anti-infective textiles. However, the recent prevalent use of nanosilver has prompted concerns for the potential adverse human and environmental effects caused from the leaching of nanosilver during usage and laundering, necessitating innovative strategies in characterizing nanosilver on textile substrates. This study investigated the viability of surface-enhanced Raman spectroscopy (SERS) as an analytical method for the characterization and quantification of nanosilver (silver nanoparticles, 12.4 ± 5.1 nm) in cotton. Two indicators, iron (III) tris (dimethyldithiocarbamate) (ferbam) and rhodamine 6G (R6G) were used to compare how efficiently they bind onto nanosilver incorporated into cotton (nanosilver-cotton) and exhibit signature SERS responses. Comparing the SERS spectra of the two indicators, R6G was found to be more appropriate for nanosilver-cotton. The solvent system for R6G was important in enhancing the SERS intensity in the cotton medium – the intensity obtained from water was nearly threefold greater than those from methanol and the 50:50 mixture of water and methanol. A linear correlation (R2 = 0.9761) between the intensity at 1503 cm−1 of R6G and the concentration of nanosilver in cotton was developed and was validated by analyzing the washing stability of nanosilver-cotton fabrics through simulated laundering cycles. The technique was also successfully applied to spatially resolve the distribution and aggregation of nanosilver on textile fabrics by mapping a coffee-ring deposition produced by the surface application of nanosilver to textile samples of raw cotton, scoured raw cotton, and 50:50 and 80:20 blends of polyester-cotton. These results validate SERS as a rapid and facile tool for monitoring the dispersion and concentration of nanosilver on textiles after application and laundering.