This study introduces a new class of eco-friendly, mineral-specific flotation collectors derived from benign biomolecules, aiming to replace conventional xanthate reagents in sulfide ore processing. We synthesized and systematically evaluated double-headed collector molecules (di-functional surfactants) for their selectivity and performance on key iron sulfide minerals (pyrite, chalcopyrite, pyrrhotite). Flotation experiments were conducted under varying collector dosages and pH conditions, complemented by zeta-potential measurements and UV–Vis spectroscopic analysis to quantify collector adsorption. We also benchmarked the new bio-based collectors against a traditional xanthate on both fresh and aged tailings to assess performance on surfaces potentially altered by storage. The results demonstrate that the novel collectors achieve comparable or superior flotation recoveries to xanthate, with markedly enhanced selectivity for target minerals. In particular, the malonate-based collector outperforms xanthate on aged pyritic materials, maintaining higher copper recovery and concentrate grade where xanthate efficiency deteriorates. These findings underscore the potential of biomolecule-derived, double-headed collectors as high-performance and environmentally sustainable alternatives in mineral flotation. The proposed reagents offer strong collecting power and selective adsorption on specific mineral surfaces while producing fewer toxic byproducts, thereby paving the way for “greener” froth flotation operations in the mining industry.