In this research, the effect of different sources of process water on the flotation efficiency of copper sulfide ore prepared from the Sarcheshmeh copper mine was investigated. For this purpose, samples of fresh water to the plant, overflows of copper-molybdenum concentrate thickener, copper concentrate thickener, and recycled water pool as well as a mixture of fresh water and recycled water were prepared and characterized. Flotation tests were performed under the same conditions as the plant’s rougher circuit and were kept constant during all experiments. Grade and recovery of copper, iron, molybdenum, and silica were selected as the metallurgical response of flotation tests. The results were subjected to statistical analysis to assess the relative significance of which water source affects the flotation performance as evaluated from the experimental results. The results showed that the copper concentrate thickener overflow had the greatest effect on the flotation efficiency, so the grade and recovery decreased by about 10% and 75% for copper, and 10% and 6% for iron in the concentrate, respectively, while the grade and recovery increased up to 0.1% and 12% for silica, and 3% and 25% for molybdenum, respectively. The reason for this effect was attributed to the high content of suspended solid particles, and Cu2+, Mo2+, and Fe2+ cations in this water source that increased the coating effect over gangue minerals and entrainment rate. The improvement of molybdenum flotation was also ascribed to the possible presence of residual diesel oil from the flotation process in the plant. Due to the relatively equal amount in all sources of process water, the effect of anions and ions of dissolved salts was difficult.