Polyurethane (PU) is a versatile elastomer that is commonly used in biomedical applications. In turn, materials derived from nanotechnology, specifically carbon nanofibers (CNFs), have received increasing attention for their potential use in biomedical applications. Recent studies have shown that the dispersion of CNFs in PU significantly enhances composite nanoscale surface roughness, tensile properties, and thermal stability. Although there have been studies concerning normal primary cell functions on such nanocomposites, there have been few studies detailing cancer cell responses. Since many patients who require bladder transplants have suffered from bladder cancer, the ideal bladder prosthetic material should not only promote normal primary human urothelial cell (HUC) function, but also inhibit the return of bladder cancerous cell activity. This study examined the correlation between transitional (UMUC) and squamous (or SCaBER) urothelial carcinoma cells and HUC on PU:CNF nanocomposites of varying PU and CNF weight ratios (from pure PU to 4:1 [PU:CNF volume ratios], 2:1, 1:1, 1:2, and 1:4 composites to pure CNF). Composites were characterized for mechanical properties, wettability, surface roughness, and chemical composition by atomic force microscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy, and goniometry. The adhesion and proliferation of UMUC and SCaBER cancer cells were assessed by MTS assays. Cellular responses were further quantified by measuring the amounts of nuclear mitotic protein 22 (NMP-22), vascular endothelial growth factor (VEGF), and tumor necrosis factor alpha. Results demonstrated that both UMUC and SCaBER cell proliferation rates decreased over time on substrates with increased CNF in PU. In addition, with the exception of VEGF from UMUC (which was the same across all materials), composites containing the most CNF activated cancer cells (UMUC and SCaBER) the least, as shown by their decreased expression of NMP-22, tumor necrosis factor alpha, and VEGF. Moreover, the adhesion of HUC increased on composites containing more CNF than PU. Overall levels of NMP-22 were significantly lower in HUC than in cancerous UMUC and SCaBER cells on PU:CNF composites. Thus, this study provided a novel nanocomposite consisting of CNF and PU that should be further studied for inhibiting the return of cancerous bladder tissue and for promoting normal non-cancerous bladder tissue formation.