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Propolis is a natural product proved to be efficient against Mycobacterium tuberculosis . Although it is produced by bees, its active alcoholic-aqueous fraction contains plant-derived molecules. To gain some insight into its mechanism of antimycobacterial activity, we studied the metabolic changes in bacterial cells treated with extract of Trigona sp. propolis from Nepal. The detailed metabolomic and transcriptomic analysis performed in this study indicated target points in bacterial cells under propolis extract influence. The profile of lipids forming the outer and middle layer of the mycobacterial cell envelope was not changed by propolis treatment, however, fluctuations in the profiles of amphipathic glycerophospholipids were observed. The enrichment analysis revealed bacterial metabolic pathways affected by Trigona sp. propolis treatment. The early metabolic response involved much more pathways than observed after 48 h of incubation, however, the highest enrichment ratio was observed after 48 h, indicating the long-lasting influence of propolis. The early bacterial response was related to the increased demand for energy and upregulation of molecules involved in the formation of the cell membrane. The transcriptomic analysis confirmed that bacteria also suffered from oxidative stress, which was more pronounced on the second day of exposure. This was the first attempt to explain the action of Nepalese propolis extract against mycobacteria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sawicki, Widelski, Okińczyc, Truszkiewicz, Glous and Sieniawska.)