Pefloxacin (PFLX) is an antibiotic, which shows broad spectrum antimicrobial activities. It is an important derivative of fluoroquinolones (FLQs) group. Ultraviolet radiation (200-400nm) causes major problem for living being which comes at the earth surface naturally through sunlight and increasing regularly due to ozone depletion. PFLX was photodegraded in 5h and forms photoproduct under UVA exposure. At the non photocytotoxic dose PFLX, shows reduced phagocytosis activity, NO (nitric oxide) production, large vacuole formation and down regulated IL-6, TNF-α and IL-1 in BALB/c macrophages at both genes and proteins levels. At higher doses (photocytotoxic doses), PFLX induced a concentration dependent decrease in cell viability of human keratinocyte cell line (HaCaT) and peritoneal macrophages of BALB/c mice. Our molecular docking suggests that PFLX binds only to the cleaved DNA in the DNA-human TOP2A complex. Topoisomerase assay confirmed that PFLX inhibits human topoisomerase by forming an adduct with DNA. Photosensitized PFLX also caused intracellular ROS mediated DNA damage and formation of micronuclei and cyclobutane pyrimidine dimers (CPDs). Increase intracellular ROS leads to apoptosis which was proved through lysosomal destabilization and reduced mitochondrial membrane potential (MMP). Our present study shows that ambient UVA exposure in the presence of PFLX caused immunomodulatory as well as photogenotoxic effects. Therefore, patients under PFLX drug treatment should avoid sunlight exposure, especially during peak hours for their photosafety.