Present study reports synthesis of Cd-free core-only and core-shell quantum dots (ZnSe of size 3.60 ± 0.12 nm and ZnSe@ZnS of size 4.80 ± 0.20 nm) having excellent fluorescent properties, stability, and aqueous solubility. The fluorescence behavior of these quantum dots (QDs) was utilized for cancer cell imaging and their comparative toxicity in cancerous (HeLa) and normal (HEK-293) cell lines was evaluated. The LC50 parameter of ZnSe (1.8 and 2.6 mg/ml) was significantly lower than that of ZnSe@ZnS (3.8 and 4.5 mg/ml) for the aforesaid cell lines which indicated higher toxicity of the core-only QD in comparison to the core-shell structure. Further, the cellular uptake and fluorescence intensity of core-shell QD was significantly higher. Furthermore, the antimicrobial property of both type of QDs was evaluated on microbes (Escherichia coli and Staphylococcus aureus) and core-shell structure was found to possess higher antimicrobial property towards gram-positive bacteria S. aureus, but was non-toxic to E. coli. These results suggested that surface modification of ZnSe with ZnS shell helps to enhance the fluorescence property and make these more biocompatible for biomedical applications. The non-toxicity towards E. coli also makes it suitable for labeling the microbial cell surface and for mapping the cellular metabolism.