Electrospun nanofibrous mats consisting of chitosan (CS) and polyvinylpyrrolidone (PVP) were constructed. Tuning of solution and process parameters was performed and resulted in an electrospun system containing a 6:4 ratio of PVP:CS. This is a significant increase in the proportion of spun CS on the previously reported highest ratio PVP:CS blend. SEM analysis showed that the nanofibrous mats with 4 wt% CS/6 wt% PVP (sample E) comprised homogenous, uniform fibres with an average diameter of 0.569 μm. XPS analysis showed that the surface of the samples consisted of PVP. Raman and FTIR analysis revealed intermolecular interactions (via H-bonding) between PVP and CS. In FTIR spectra, the contribution of chitosan to CS/PVP complexes was shown by the downshift of the C=O band and by the linear increase in intensity of C-O stretching in CS. XPS analysis showed a smaller shift at the binding energy 531 eV, which relates to the amide of the acetylated functional groups. The obtained results demonstrate a sensitivity of Raman and FTIR tests to the presence of chitosan in PVP:CS blend. The chemotherapy drug 5-Fu was incorporated into the constructs and cell viability studies were performed. WST-8 viability assay showed that exposure of A549 human alveolar basal epithelial cells to 10 mg/mL 5-Fu loaded fibres was most effective at killing cells over 24 h. On the other hand, the constructs with loading of 1 mg/mL of drug were not efficient at killing A549 human alveolar basal epithelial cells. This study showed that CS/PVP/5-Fu constructs have potential in chemotherapeutic drug delivery systems.