Background: Bone marrow-derived mesenchymal stem cells (BMSCs) are ideal candidates for cell-based therapy due to their self-renewal and multilineage differentiation ability. During in vitro expansion, BMSCs tend to lose their proliferation rate and multipotency limiting their clinical use. Hence, supplementation of basic fibroblast growth factor (bFGF) during in vitro culture might positively influence the biological and phenotypic properties of expanded BMSCs. Aim: This study was aimed to evaluate the supplementation of bFGF on selected biological and phenotypic characteristics of BMSCs. Methods: Plastic-adherent BMSCs were cultured without (0 ng/mL) and with (5 ng/mL and 10 ng/mL) bFGF up to five passages and analyzed for their morphology, viability, proliferation rate, population doubling time (PDT), colony forming unit-fibroblast (CFU-F) assay, senescence activity, genetic stability and cell surface marker expression. Results: BMSCSs exhibited a small, spindle-shape morphology in bFGF supplemented groups as compared to elongated fibroblast-like cells in control. No significant difference in viability and PDT was observed. However, noticeable differences were observed in proliferation rate and CFU-F ability between bFGF supplemented group and control. Further, the senescence activity of BMSCs was considerably decreased under the influence of bFGF. BMSCs had a normal karyotypein both bFGF supplemented and control groups. Lastly, addition of bFGF in expansion media slightly modified the phenotypic markers expression in BMSCs, corresponding to the concentrations used. Conclusion: Supplementation of BFGFat10 ng/mL could be considered as an optimal and efficient concentration for expanding BMSCs in culture before their use in cell-based therapy.