Over the past few decade, thiolate-protected metal nanoclusters have gained great attention due to their high surface-to-volume ratio and greater surface area which finds applications in electronic, optical, magnetic, and biomedical fields. Herein, we report the tailoring of surface ligands protected over the ultra-small iron nanoclusters using three different thiols namely glutathione (GSH), 3-mercaptopropionic acid (MPA) and 3-mercaptopropane sulfonate (MPS). To understand the role of surface ligands on as prepared FeNCs (namely FeGSH, FeMPA and FeMPS NCs) was characterized by photophysical and bioimaging studies. The HRTEM and AFM results confirmed that these FeGSH, FeMPA and FeMPS NCs were in the average size range of 1–6 nm. The varying functionality and robustness of GSH, MPA and MPS protected over these individual FeNCs resulted in tunable quantum yields of 5.6 %, 5.8 % and 2.1 % for FeGSH, FeMPA and FeMPS NCs respectively. Further, these three FeNCs were investigated for cell viability and fluorescent microscopic analysis with the MCF-7 breast cancer cell line. The cell viability of FeGSH, FeMPA and FeMPS NCs with cancer cell line were found to be 96.62 %, 98.31 % and 93.29 % respectively for a fixed concentration range of 0 to 100 nM. The Phase contrast and fluorescent microscopy results confirm FeNCs induction which demonstrates that the cells were noncytotoxic and biocompatible for various applications, and this was further validated by the MTT test. The in vitro results imply that the ligand shell thickness and functionality here resulted in varying bioimaging results and antibacterial properties.