Plant-derived natural products, including those from bryophytes, are particularly advantageous in several aspects, as for antifungal, antibacterial, and anticancer properties. However, the bioactivity potential of secondary metabolites found in the liverwort species Acrolejeunea fertilis (A. fertilis) has not been extensively investigated; moreover, it has limited biomass in nature. An in vitro culture can be a solution for the propagation of A. fertilis. The application of plant growth regulatory substances with various concentrations is expected to increase the growth of A. fertilis. This study aimed to determine the optimum concentration of 2,4-dichlorophenoxyacetic acid and kinetin applied to the gametophyte phase of A. fertilis in vitro culture. This study also aimed to compare the metabolite profile between A. fertilis from nature and in vitro culture condition, using methanol and n-hexane solvent. The compounds in the extracts were identified using gas chromatography-mass spectrometry (GC-MS) also their antioxidant and antibacterial activities. 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. The antibacterial activity revealed inhibition on S. aureus using an n-hexane A. fertilis from nature. Thus, this study highlights the diverse secondary metabolite profiles and bioactivity potential of A. fertilis extracts. These findings have important implications for the development of natural products for medicinal and mass propagation in terms of the potential of A. fertilis as a source of bioactive compounds.