Abstarct: Flotation is the most important method for processing sulfide copper ores. Due to the high cost and environmental hazards caused by the chemical reagents used in this process (such as collectors, frothers, pH regulators, and depressants), the possibility of replacing all these reagents or at least some of them are of special importance through environmentally friendly methods such as biotechnology. One of these methods is bioflotation using halophilic bacteria. In this research, three stages of testing were conducted. In the first stage, five types of halophilic bacteria were studied for pyrite biodepression and chalcopyrite flotation. Bioflotation experiments were conducted using Hallimond tubes, and the bacteria Halobacillus sp., Alkalibacillus almallahensis, and Alkalibacillus sp. had better performance in pyrite depression and chalcopyrite flotation than other bacteria. The recovery of pyrite depression when using them was 30.9, 30.3, and 34.0 %, respectively, and the recovery of chalcopyrite flotation by them was equal to 52.9, 68.6, and 55.7, respectively, which indicates the high selectivity of these bacteria in flotation. In the second stage, the effect of the combination of these three types of bacteria on pyrite depression and chalcopyrite flotation was also studied. The results indicated that in the combination (mix) test of all three types of bacteria (33.3% of each type), pyrite was depressed better than other tests, and its recovery was 27.5%, which was lower than the single bacteria tests. Also, the effect of the combination of these three types of bacteria on the flotation of chalcopyrite was investigated and its recovery was 72.6%, which was higher than the single bacteria tests. On the other hand, considering that the recovery of chalcopyrite in the three-bacteria combination tests was higher than the single-bacteria and two-bacteria tests, it can be concluded that the combination of all three bacteria can cause better synergism and improve their performance in microflotation tests. In the third stage, four types of halophilic bacteria Halobacillus sp., Alkalibacillus almallahensis, Marinobacter sp., and Alkalibacillus sp. were studied to replace frothers (MIBC and F7240), depressant (gasoline), and pH regulator (lime) in sulfide copper flotation using a Denver laboratory flotation cell. It is worth mentioning that it was not possible to replace collectors (Z11 and C7240) with these bacteria because halophilic bacteria play the role of depressants. On the one hand, due to their growth in a neutral medium, it is not necessary to use pH regulators such as lime along with them. On the other hand, used frothers cause the death of bacteria. Therefore, they are only used together with collectors. The results indicated that each of the four types of bacteria mentioned above along with the mentioned collectors as the only chemical reagents (bioflotation + collector) can depress pyrite better than the bacteria-free mode (flotation + all chemical reagents). However, the recovery of chalcopyrite using the bioflotation method was lower than its recovery using the flotation method. Pyrite depression recovery in flotation method was 63.6% which was respectively reduced to 5.7, 22.0, 28.5, and 20.8% by using halophilic bacteria of Halobacillus sp., Alkalibacillus almallahensis, Marinobacter sp., and Alkalibacillus sp.. On the other hand, chalcopyrite flotation recovery in the flotation method was 89.4%, which reached 54.8%, 69.7%, 55.1%, and 67.4%, respectively, using the above bacteria in the bioflotation method.