In recent decade use of microorganisms and their metabolites for selective separation of minerals, especially in bio-flotation and bio-flocculation methods, has received increasing attention by researchers. Compared to the conventional chemical agents, bio-reagents with environmental friendliness nature, non-toxic and bio-degradable, non-destructiveness influence on physical and chemical structure of the minerals and lower costs, have been able to provide promising results in mineral separation. Therefore, in this study, the application of cell and produced metabolite of the Bacillus licheniformis bacteria, as a bio-collector in the flotation, and bio-flocculant in the sedimentation of barite and silica minerals, with the aim of proper selective separation of these two minerals were investigated. Flotation and settling experiments were carried out to obtain the optimum separation conditions through evaluation of the effect of, bio-surfactant type, culture medium and suspension pH, bacteria cells and minerals contact time, bacteria cells concentration and process time. The zero point of charge for barite, silica and bacterial loads were determined to occur at pH of 6.3, 2.8 and 2, respectively. Measurements showed that, the cell adsorption decreases by increasing solution pH, and the highest adsorption concentration for the barite is achievable at pH = 3, and was about 1.33 ×1010 cfu/g. Based on the conducted floatation experiments assisted by various bio-surfactants in a modified Hallimond tube, and under the optimum conditions, the maximum recovery of the barite and silica to the float section was determined to be about 87% and 15% under the solution pH of 3, flotation time of 3 minutes and after pre cell-mineral interaction (each mineral separately). The flotation quality of minerals in interaction with pure medium and medium containing bacterial metabolites were not satisfactory and the results were very similar to the control tests. After investigating the effect of various parameters and optimizing each of them by single mineral experiments, and to evaluate the feasibility of the bio-flotation method for selective separation of barite from silica, flotation experiments were performed using a sample of 1:1 mixture of barite and silica under the determined optimum conditions for solo minerals. The results showed that the best separation of barite from silica is obtainable after the interaction of the mixed sample with the suspension of 6.55×103 cfu/ml bacteria cells at pH=3. After 3 min of flotation, 76% and 4% of the barite and silica, respectively, were collected as the floating particles. Increase of flotation time (up to 7 minutes) had no significant improving effect on selective separation of the minerals. Further experiments were carried out to investigate the selective separation of barite and silica through settling method. After investigating the effect of various factors on the particles deposition in the suspension, the maximum amount of separation under optimum conditions, under interaction of both minerals with bacteria cells at pH of 3 and 11, and 3 min settling time, were achieved to be 92% and 98% for barite, respectively, and 66% and 98% for silica.