In this study, stable isotopes of carbon (δ 13 C), sulfur (δ 34 S), oxygen (δ 18 O), hydrogen (δ 2 H), nitrogen (δ 15 N) and radioactive isotope of hydrogen (tritium) have been applied in combination with conventional techniques (physiochemical and biological) to investigate Karachi Coastal water pollution due to Layari and Malir rivers, which mainly carry Karachi Metropolitan domestic and industrial wastewater, and to determine the effect of seawater on the aquifer system of the area. Physico-chemical (pH, electrical conductivity and turbidity), biological (fecal coliform), heavy metal and stable isotope (carbon and sulfur) characteristics of Layari and Malir rivers water, Karachi coastal waters and sea sediments showed that Karachi Metropolitan domestic sewage and industrial effluents were the main source of coastal water pollution, and coastal water of Manora Channel was heavily polluted as compared to water of southeast coastal area of Karachi. However, the northwest coastal water was marginally polluted. Turbidity and fecal coliform population levels in seawater of Manora Channel and some locations along southeast coast (Ghizri area and Ibrahim Haideri) were found above the WHO recommended threshold limits set for seawater bathing. Heavy metal contents of Manora Channel and southeast coastal waters were higher than the Swedish guidelines for the quality of seawater. In contrary, heavy metal concentrations in coastal sediments were found to be significantly higher than that of seawater. Mn and Ni contents in sediments of entire coast (Manora Channel, southeast and northwest coast) were above USEPA guidelines except at Buleji site, whereas Cr, Zn and Cu levels only in Manora Channel sediments were higher than USEPA guidelines. The higher heavy metal contents of Manora Channel water and sediments can be attributed to an influx of a major portion of untreated industrial and/or domestic wastewater. Layari and Malir rivers water was observed to be depleted in δ 13 C (TDIC) and δ 34 S which showed heavy influx of sewage into these rivers. Manora Channel water was also depleted in δ 13 C TDIC and δ 34 S during low tide environment showing a large scale domestic wastewater mixing with seawater. Southeast coastal water was found to be slightly enriched in δ 13 C (TDIC) and δ 34 S and exhibited mixing of relatively small quantity of sewage with the seawater as compared to Manora channel. δ 13 C (TDIC) and δ 34 S contents of northwest coastal water were close to the values meant for normal seawater. δ 13 C and δ 15 N contents of Karachi coastal seaweed ranged from -31.1 to -4.9 ‰ PDB and 6.1 to 17.8 ‰ Air, respectively. Average δ 15 N values (10.2 ‰ Air) of Ulva spp. collected from non polluted northwest coast was higher as compared to average δ 15 N contents (8.0 ‰ Air) of Ulva fromii Manora Channel suggesting that nitrogen isotopic ratios of Ulva spp. could be a good indicator of sewage pollution. The results of a two component isotope mass balance equation using δ 13 C and δ 34 S values for Layari and Malir rivers and coastal water indicated that tide conditions and distance of sampling site from the pollution source were the main factors to control transport and dissemination of Layari river pollution into Manora Channel. High tide environment slowed down the Layari river water mixing with seawater coupled with a gradual decrease in pollution levels from Layari river out fall zone to Manora Lighthouse. Physico-chemical characteristics of groundwater showed that shallow aquifer system was not generally affected by seawater intrusion. Deep groundwater was observed to be moderately (chloride contents 1000- 2000 ppm) to highly saline (chloride contents > 2000 ppm). However, shallow and deep well located in vicinity of the Malir and Layari rivers showed fecal coliform contamination which rendered their water unfit for drinking. An anion based piper diagram showed predomination of chloride and bicarbonate ions relative to sulfate (SO 4-2 ) ions in coastal groundwater. It was inferred from Piper diagram that shallow aquifer system was predominantly replenished by freshwater (mainly Layari and Malir rivers basin), while salinity of deep aquifer owes to the seawater intrusion or entrapped salinity. The δ 18 O composition of groundwater and δ 2 H vs. δ 18 O relationship for groundwater of coastal aquifer also indicated that shallow aquifer system was predominantly recharged through fresh water sources. Salinity of deep aquifer seemed to originate from seawater intrusion and/or from entrapped salinity. These finding were strongly endorsed by anion and stable isotope interpretations such as Cl - concentration vs δ 18 O, Cl - /HCO 3-1 ratio vs δ 18 O values and SO 4-2 contents against SO 4-2 /Cl - ratio of shallow and deep aquifers. The present study has demonstrated that stable isotopes (δ 13 C, δ 15 N, δ 34 S, δ 2 H and δ 18 O) can be effectively used to monitor the marine pollution and to investigate origin of salinity in the coastal aquifer. This study may also provide a precise and accurate isotopic database for researchers interested in seawater pollution and its effect on benthic life.