The information available on microalgae-sourced compounds, especially antibiotics and other bioactive compounds, and their potential commercial applications is still insufficient. In this study, antibacterial activity, metabolites, and molecular characterization of Phormidium autumnale, which was isolated from samples collected from different natural freshwater sources in Ankara, Turkey, were investigated. Sequencing results of 16s rDNA confirmed the molecular identification of P. autumnale by 99%. It was determined that the peak values of some phenolic compounds and cyclic peptides were consistent with the 1653-1389 cm(-1) band regions in the FTIR spectra of the species. The antibacterial activities of P. autumnale cyanobacteria (CBA) extracts that were obtained by using different solvents were tested on Escherichia coli, Staphylococcus epidermidis, methicillin-resistant (MR) Staphylococcus aureus, Streptococcus agalactiae, and Enterococcus faecalis by using a disc diffusion method. Also, the minimum inhibition concentration (MIC) and antimicrobial indexes of all extracts were determined. It was found that P. autumnale methanol extracts showed antibacterial activity on all test bacteria, whereas acetone extracts showed effects only on E. coli. For the inhibition of MR S. aureus, the control methanol extract was found to give very similar results to those exhibited by the control antibiotics, and the antimicrobial index results were determined to be 58.7-67.5%. According to the results of the analysis of methanol extract, gentisic acid, vanillic acid, 4-hydroxybenzoic acid, p-coumaric acid, and catechin (especially phenolic compounds) were determined to be the active compounds. It can be concluded that P. autumnale is an alternative to current commercial applications as an antibacterial agent in phytotherapy.