Harike is a wetland of international importance under the Ramsar Convention. The present study entails the investigation of mutagenic, genotoxic and cytotoxic effect of surface water samples collected from five different areas of the Harike wetland by using the histidine reversion point mutation assay in Salmonella typhimurium (TA98 and TA100) strain with or without S9, bioluminescence mutagenicity assay using Vibrio harveyi (A16) strain, plasmid-nicking assay using pBR322 and 3(4,5-dimethyl-thiazol-2-yl)2,5-diphenyl-tetrazolium bromide assay as well as confocal imaging studies using Chinese hamster ovarian cell line, respectively. It was observed that although, the water sample of all the areas of wetland demonstrated mutagenic, genotoxic as well as cytotoxic activity, the effect was quite significant with the water samples from River Satluj and Khatan area (i.e. reservoir mainly contains Satluj water). The metal analysis of water samples was also conducted with atomic absorption spectrophotometer. The mutagenicity, genotoxicity and cytotoxicity of water samples emerged to be correlated with metal concentration. The source of toxic components seems to be associated with various industrial effluents and agricultural run-off. The results of the present study carry great importance in documenting the water quality monitoring data of the wetland.