Froth flotation is the most preferred processing technique for the enrichment of low-grade sulfides. Bioleaching is an eco-friendly method for metallurgical extraction from flotation products. Flotation reagents (collectors, frothers, etc.) have various impacts on bioleaching and bacterial activities. In this investigation, the effect of a number of sulfide flotation collectors [potassium amyl-xanthate, potassium isobutyl-xanthate, sodium ethyl-xanthate, potassium isopropyl-xanthate, and Dithiophosphate (Aero3477)], and frothers (pine oil and methyl isobutyl carbinol) with different dosages is studied on Leptospirillum ferrooxidans activities. The results of various measurements indicated that these flotation chemicals can have positive or negative influences on the bacterial activities, based on their chemical compositions and/or concentrations. These results can extensively be used for the selection of flotation reagents when bioleaching is chosen as the metallurgical extraction method after flotation enrichment.