In the current study, the occurrence of different microorganisms in a microcosm experiment of polluted River Nile water with different concentrations of chlorpyrifos insecticide was estimated. The obtained results showed the presence of twenty-three fungal species related to nine fungal genera. Aspergillus, Penicillium, Fusarium and Trichoderma were the most prevalent fungal genera. The highest occurrence was recorded for Trichoderma sp. at 0.05 mL/L chlorpyrifos and the lowest occurrence was recorded for Botrydiplodia and Curvularia at control microcosm experiment and Stachybotrys at 0.2 mL/L chlorpyrifos. The highest fungal taxa (S) and individuals were estimated in the control experiment and the lowest fungal taxa and individuals were assessed at 0.1 mL/L chlorpyrifos. Whereas, the highest fungal Dominance (D) was monitored at 0.1 mL/L chlorpyrifos and the lowest fungal dominance (D) index was recorded at 0.05 mL/L chlorpyrifos insecticide. So, the highest fungal biodiversity indexes of Simpson (1-D) and Shannon (H) were estimated at 0.05 mL/L concentration of chlorpyrifos insecticide and the lowest fungal Simpson and Shannon indexes were recorded at 0.1 mL/L concentration. From the correlation results between the physicochemical characteristics and fungal occurrence, it was found that Aspergillus fumigatus was impacted by both the temperature and total dissolved sulphates (TDS), whereas Penicillium duclauxii was affected by pH, conductivity, and total salts (Ts), and Aspergillus niger was influenced by both total dissolved carbon (TDC) and total dissolved nitrogen (TDN). The cluster analysis of fungal composition estimated that fungal groups were detected with different concentrations of insecticide chlorpyrifos.