Plant-derived natural products, including those from bryophytes, are particularly advantageous in several aspects, such as for their antifungal, antibacterial, and anticancer properties. However, the bioactivity potential of secondary metabolites found in the liverwort species Acrolejeunea fertilis has not been extensively investigated, with its limited natural biomass posing challenges. A possible solution to address this is to propagate A. fertilis via in vitro culture. The application of plant growth regulatory substances at various concentrations is expected to enhance the growth of A. fertilis. This study aims to determine the optimum concentration of 2,4-dichlorophenoxyacetic acid and kinetin applied in the gametophyte phase of A. fertilis in vitro culture and compare the metabolite profile between A. fertilis from nature and in vitro culture conditions, using methanol and n-hexane solvent. The compounds in the extracts were identified using gas chromatography–mass spectrometry (GC-MS), and their antioxidant and antibacterial activities were also characterized. The IC50 values for nature and in vitro extracts were in the range of 58.41–63.10 µg/mL and 111.73–112.10 µg/mL. The total value of phenolic compounds in A. fertilis extracts ranged from 58.51±0.02 µgGAE/g and 272.53±0.02 µgQE/g, respectively. Antibacterial activity was revealed, with inhibition on S. aureus using an n-hexane extract of A. fertilis from nature. Thus, the findings of this study highlight the diverse secondary metabolite profiles and bioactivity potential of A. fertilis extracts. These findings have important implications for the development of natural products in terms of the potential for mass propagation of the medicinal plant A. fertilis as a source of bioactive compounds. © The Author(s), under exclusive licence to Springer Nature B.V. 2024.