Biocompatible polymers have received significant interest from researchers for their potential in diagnostic applications. This type of polymer can perform with an appropriate host response or carrier for a specific purpose. The current study aims to fabricate and characterise poly(ethylene) oxide (PEO) nanofibres with different concentrations for cytotoxicity evaluation in human breast cancer cell lines (MCF-7) and to get an optimal PEO nanofibre concentration (permissible limit) as a suitable polymer matrix or carrier with potential use in diagnostic applications. The fabrication of PEO nanofibres was done using electrospinning and was characterised by structure and morphology, surface roughness, chemical bonding and release profiles. The functional effects of PEO nanofibres were evaluated with MTS assay and colony formation assay in MCF-7 cells. The results showed that viscosity plays a vital role in synthesising a polymer solution in electrospinning for producing beadless nanofibrous mats ranging from 4.7 Pa·s to 77.7 Pa·s. As the PEO concentration increases, the nanofibre diameter and thickness will increase, but the surface roughness will be decreased. The average fibre diameter for 5 wt% PEO, 6 wt% PEO and 7 wt% PEO nanofibres were 129 ± 70 nm, 185 ± 55 nm and 192 ± 53 nm, respectively. In addition, the fibre thickness for 4 wt% PEO, 5 wt% PEO, 6 wt% PEO and 7 wt% PEO nanofibres were 269 ± 3 µm, 664 ± 4 µm, 758 ± 7 µm and 1329 ± 44 µm, respectively. Contrarily, the surface roughness for 4 wt% PEO, 5 wt% PEO, 6 wt% PEO and 7 wt% PEO nanofibres were 55.6 ± 9 nm, 42.8 ± 6 nm, 42.7 ± 7 nm and 36.6 ± 1 nm, respectively. PEO nanofibres showed the same burst release pattern and rate due to the same molecular weight of PEO with a stable release rate profile after 15 min. It also demonstrates that the percentage of PEO nanofibre release increased with the increasing PEO concentration due to the fibre diameter and thickness. The findings showed that all PEO nanofibres formulations were non-toxic to MCF-7 cells. It is suggested that 5 wt% PEO nanofibre exhibited non-cytotoxic characteristics by maintaining the cell viability from dose 0-1000 µg/ml and did not induce the number of colonies. Therefore, 5 wt% PEO nanofibre is the optimal nanofibre concentration and was suggested as a suitable base polymer matrix or carrier with potential use for diagnostic purposes. The findings in this study have demonstrated the influence of cell growth and viability, including the effects of PEO nanofibre formulations on cancer progress characteristics to achieve a permissible PEO nanofibre concentration limit that can be a benchmark in medical applications, particularly diagnostic applications.