The fact that froth flotation consumes a large amount of water, even though it is one of the most efficient methods to process sulfide minerals, brings a higher pressure from the sustainable development due to increasing population and its demand of more portable water. Pioneering work of applying secondary effluent in sulfide flotation (Fisher and Rudy, 1976) showed a 2.4% reduction in Cu recovery and 16.2% reduction in molybdenum recovery when secondary effluent was used. It was also postulated that the organic carbon, in the form of humic acid, in the effluent was the most deleterious constituent causing the losses in metal recovery. We carried out a systemic investigation in both microscopic and macroscopic aspects on the possibility of using secondary effluent in sulfide flotation. AFM images showed that collectors adsorbed on mineral surface (chalcopyrite and molybdenite) in a similar manner in both clean water and treated secondary effluent. Lab flotation tests showed that the Cu and Mo recoveries obtained with treated secondary effluent were comparable to those obtained with tap water. The findings of present multi-scale investigation will help provide a cost-efficient solution to treat low quality water and mitigate its impact, and finally succeed technically in applying secondary effluent in sulfide flotation.