Pollutants cause a huge problem for humans, animals, plants, and various ecosystems, especially water resources. Agricultural, domestic, and industrial waste effluents change the water quality and affect living microorganisms. Therefore, the current study aimed to identify possible microorganisms in wastewater as potential bioremediation agents of pesticide residues. Wastewater samples were collected from El-Khairy agricultural drainage, which receives agricultural and domestic wastes. Bacteria and fungi species were isolated as clean cultures. Wastewater samples were analyzed for pesticide residues via gas chromatography-mass spectroscopy (GC–MS) system. Results uncovered the presence of ten pesticides ranging from 0.0817 to 28.162 µg/l, and the predominant pesticide was chlorpyrifos. Along with that, about nine species (3 bacterial and 6 fungal) were relatively efficient in the removal of chlorpyrifos residues up to 2000 µg/l with removal percentages ranging from 24.16 to 80.93% under laboratory conditions. Two bacterial isolates proficiently degraded significant amounts of chlorpyrifos:Bacillus cereusstrain PC2 (GenBank accession No. MZ314010) andStreptomyces praecoxstrain SP1 (GenBank accession No. MZ314009). In-site bacterial and fungal isolates defined in the current study were proficient in cleaning wastewater of chlorpyrifos pesticide residues.