The need for new antimicrobial agents is greater than ever because of the emergence of multidrug resistance in common pathogens and incidence of new infections. Cyclopent-4-ene-1,3-diones (CPDs) have been reported as a new class of compounds with promising antimicrobial and antifungal properties. Herein we report the selective antibiotic properties of nostotrebin 6, a phenolic CPD produced biotechnologically by the culture of cyanobacterium Nostoc sp. str. Lukešová 27/97. High performance countercurrent chromatography (HPCCC) combined with gel permeation chromatography (GPC) was used for the isolation of nostotrebin 6 with a relatively high 0.53 ± 0.1% yield (calculated from dried biomass) and final purity higher than 96%. Nostotrebin 6 was tested for its antimicrobial and antifungal activities by using standard micro-dilution method, and the results were expressed as minimal inhibitory concentrations (MICs). Nostotrebin 6 unequivocally inhibited the growth of Gram-positive reference (Enterococcus faecalis CCM 4224, Staphylococcus aureus CCM 4223 and Staphylococcus aureus CCM 3953) and multidrug-resistant (Staphylococcus haemolyticus A/16568, Staphylococcus aureus MRSA 4591 and Enterococcus faecium VanA 419/ana) strains. Its strongest effect was exerted against the Gram-positive bacteria with MICs ranging between 6.25 and 15.6 μg/mL. There was no effect on Gram-negative strains tested and yeasts. Our results suggest that nostotrebin 6 could serve as basic nucleus for further design of novel antibiotic agents and demonstrate that the bio-production approach based on HPCCC/GPC isolation endpoint is an efficient methodology for obtaining nostotrebin 6 in multi-gram scale. Furthermore, the presented isolation method can be easily up-scaled to process kilograms of the cyanobacterial biomass.