Effective pyrite depression is required to improve the grade of copper concentrate from a flotation process. Raw seawater can be used directly in a flotation plant to replace desalinated freshwater. However, seawater causes some problems and prevents efficient flotation. We aimed to effectively depress pyrite in raw seawater using marine iron-oxidizing bacteria (bio-flotation). Basic experimental conditions using four strains of bacteria were assessed in this study. Suitable cultivating conditions using FeS2 and FeCl2 as Fe sources were determined. Bacterial growth increased when FeS2 was used because Fe2+ was supplied more continually by pyrite than FeCl2 in seawater. The bacteria proliferated effectively at 25 °C, even though the temperature of the seafloor the bacteria originally inhabited was different. A flotation experiment was performed using a Hallimond tube after allowing the culture solution to react with 0.5 g pyrite (106–150 μm) for 30 min. The pyrite recovery was 10–20% lower after the pyrite had reacted with seawater containing Fe-oxidizing bacteria, but the pyrite recovery increased to 80–90% when the subculture (the original source of the culture used in the flotation experiment) period was longer using all bacterial strains. The bacterial cell concentrations in the experiments were 1 × 106–2 × 107 cells/mL. However, the pyrite recovery and bacterial cell concentration did not positively correlate. This indicated that the bacterial activity related to the subculture was an important factor promoting the bacterial reaction, i.e., by increasing hydrophilicity of the pyrite surface.