To optimise the culture conditions for human Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) intended for clinical use, we investigated ten different properties of these cells cultured under 21% (atmospheric) and 5% (physiological normoxia) oxygen concentrations. The obtained results indicate that 5% O2 has beneficial effects on the proliferation rate, clonogenicity, and slowdown of senescence of hWJ-MSCs; however, the oxygen level did not have an influence on the cell morphology, immunophenotype, or neuroprotective effect of the hWJ-MSCs. Nonetheless, the potential to differentiate into adipocytes, osteocytes, and chondrocytes was comparable under both oxygen conditions. However, spontaneous differentiation of hWJ-MSCs into neuronal lineages was observed and enhanced under atmospheric oxygen conditions. The cells relied more on mitochondrial respiration than glycolysis, regardless of the oxygen conditions. Based on these results, we can conclude that hWJ-MSCs could be effectively cultured and prepared under both oxygen conditions for cell-based therapy. However, the 5% oxygen level seemed to create a more balanced and appropriate environment for hWJ-MSCs.