Liposomal levofloxacin was prepared with the extrusion technique. Levofloxacin is a fluoroquinolone used for resistant tuberculosis treatment. Traditional lipids such as phosphatidylcholine (PC), as well as a mixture of PC and cholesterol (Chol) were used for liposome preparation. Antibiotic encapsulation into the liposomes was carried out with the passive and active loading methods. The active method of levofloxacin loading into liposomes was conducted with ammonium sulphate gradient. Levofloxacin encapsulation efficiency into liposomes containing PC/Chol (4:1, w/w) was found to be as high as 90% when active the loading method was used, while levofloxacin encapsulation efficiency was as low as 20% when the passive loading method was used. Levofloxacin releasing kinetics with Franz diffusion cell was investigated. Levofloxacin was shown to release gradually from liposomes containing PC and Chol to 18% for 21 h, while levofloxacin released completely for 3 h from antibiotic solution. The prepared liposomal levofloxacin was used to test its activity on extensively drug resistant strain Mycobacterium tuberculosis СN-37 growth with automatized system BACTEC MGIT 960. Mycobacterium tuberculosis СN-37 was resistant to rifampicin, isoniaside, streptomicin, ethambutol, pyrazinamide, ofloxacin, amikacin, capreomicin. Mycobacteria growth monitoring with automatized system BACTEC MGIT 960 was based on fluorescence measurement of fluorophore located at the bottom of Mycobacteria Growth Indicator Tube (MGIT). This fluorophore is «neutralized» by high oxygen concentrations. The living bacterial cells take in oxygen, which results in fluorescent increase. The liposomal levofloxacin as well as levofloxacin solution at 1 and 2 mkg/mL were found to delay mycobacteria growth by 1-2 days while both levofloxacin forms at 4 mkg/mL inhibited mycobacteria growth completely. It means that the liposomal levofloxacin is active as an antibiotic solution against mycobacteria. Summing up, it is the active loading method that can be rationally used for the liposomal levofloxacin preparing with maximal encapsulation efficiency. The prepared liposomes loaded with levofloxacin allow the antibiotic to release gradually. So, the antibiotic is active for a long time. It is important for increase of the antibiotic activity within human organism.