Abstract: Under the steadily intensifying human activities in coastal areas, it is crucial to distinguish the sources of organic matter (OM) in sediments for better understanding of the environmental change. In this study, 25 surface sediments collected along the harbor and creeks areas off Karachi coast were used for total organic carbon (TOC), total nitrogen (TN), stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotopic compositions, phytoplankton biomarkers (brassicasterol, dinosterol and cholesterol) and glycerol dialkyl glycerol tetraethers (GDGTs) analyses, to trace the distribution of OM and identify its sources. TOC, TN, δ¹³C, and δ¹⁵N are in the range of 0.04% to 5.28%, 0.01% to 0.57%, − 17.55‰ to − 26.31‰, and 2.83‰ to 6.04‰, respectively. C/N values range from 4.24 to 21.04. The δ¹³C, δ¹⁵N, C/N, BIT (Branched Isoprenoid Tetraether) index together with phytoplankton biomarkers show both marine and terrestrial OM input to the creeks and Harbor stations. In contrast, the stations from Western Coast indicate a marine source predominance. A clearly impact of human activities is seen from sediments near river mouths.