Sparfloxacin (SPFX) is a broad spectrum antibiotic which inhibits bacterial DNA gyrase enzyme activity. However, photodegradation in the presence of UVA limits its antibacterial activity and induces phototoxicity. Thus, to encounter this problem, we have developed poly d,l-lactic-co-glycolic acid (PLGA) loaded SPFX nanoparticles. Here, we have performed a comparative antibacterial activity of SPFX and its nanoparticles (NPs) through molecular docking and plate sensitivity assay. Under environmental UVA exposure, photoexcited SPFX significantly generates ROS, DNA damage and mitochondrial mediated cell death in comparison to PLGA-SPFX-NPs (nano SPFX) in human skin cell line (HaCaT). In presence of UVA, bulk SPFX induced cell cycle arrest with appearance of sub-G1 peak showing apoptosis while nano SPFX did not show any change. SPFX triggered apoptosis via alteration in membrane integrity of mitochondria and lysosome in comparison to PLGA-SPFX-NPs. Involvement of mitochondrial mediated cell death was confirmed by down-regulation of anti-apoptotic Bcl-2 and procaspase-3 and upregulation of pro-apoptotic Bax, cytochrome-c and caspase-3 proteins expression. Specific caspase inhibitor, Z-VAD-FMK showed involvement of caspase cascade pathway in apoptosis. Our finding suggests that controlled release of SPFX from PLGA-SPFX-NPs can reduce its side effects and enhance its antibacterial activity. Thus, nanotization of fluoroquinolones will be a significant step to reduce the problem of resistance and phototoxicity of this group.