The continuous input of petroleum-based and other industrial pollutants along with heightened navigational activities in the inland and coastal waters of the Niger Delta region of Nigeria has contributed tremendously towards petroleum pollution of the aquatic environment. Standard analytical methods were employed in sample collection and analysis. The results showed the mean values of Total Heterotrophic Bacteria [THB] for tidal water 1.44±0.20 (x107), 1.42±0.62 (x107)and 1.82±0.61 (x107)for upstream, midstream and downstream respectively, while the mean values for CUB 1.06±0.12 (x106), 1.30±0.54 (x106) and 1.28±0.46 (x106) for upstream, midstream and downstream respectively. Similarly, the mean values for Total Fungi (TF) and Crude oil-Utilizing Fungi (CUF) were 1.08±0.12 (x106), 1.12±0.21 (x106), 1.18±0.20 (x106) and 8.2±0.78 (x104), 9.2±0.20 (x104), 8.8±0.26 (x104) for upstream, midstream and downstream respectively. For intertidal water, the mean values obtained for upstream, midstream and downstream were 1.24±0.82 (x107), 1.77±0.57 (x107) and 1.40±0.32 (x107) for THB, 1.08±0.92 (x106), 1.08±0.22 (x106) and1.13±0.21 (x106) for CUB, 1.00±0.60 (x107), 1.26±0.30 (x106) and 1.11±0.18 (x106) for Total fungi [TF] and 7.2±0.81 (x104), 9.6±0.4 (x104), 9.0±0.27 (x104) for CUF). While the values for benthic sediment were 1.55±0.38 (x108), 1.68±0.32 (x108), 2.24±0.34 (x108) for THB, 1.14±0.32 (x107), 1.24±0.88 (x107), 1.48±0.90 (x107) for CUB, 1.12±0.31 (x107), 1.20±0.52 (x107), 1.40±0.16 (x107) for TF and 8.2±0.12 (x105), 6.2±0.43 (x105), 1.01±0.12 (x106) for CUF. The results showed that there was no significant difference (p> 0.05) in the mean values of each physicochemical parameter across the different microhabitats and stations. This result revealed the massive impacts of anthropogenic gradients on the biology and physicochemistry of Iko River estuary.