Generally, during drug discovery programs, after identification of new antibiotic metabolite, its high quantity production is obtained by various approaches, including production or extraction improvement or even strain genetic manipulation. To provide enough amounts of two novel non-toxic anti-MRSA pentapeptides named persipeptides (A and B) required for drug discovery programs, seven different fermentation broths examined. CM1 medium considerably enhanced the biosynthesis of persipeptides up to 219.63 ± 2.48, compared with ISP2 medium (36.31 ± 1.37), showing a six-fold increase. Additionally, at the extraction level, results of experimental design indicated that liquid-liquid extraction (LLE) of persipeptides by 34 % BuOH at 228 rpm (Stirrer speed), temperature 28 ˚C, and pH 9-9.5 for 78 min (stirring time) was equal to 264 ± 9.85 μg mL-1, which was the most favorable combination for their extraction. Compared with un-optimized extraction process (219.63 ± 2.48 μg mL-1), the optimized conditions improved the yield of the extraction by 20.20 %, while saving both time and solvent usage up to 67 % (162 min) and 16 %, respectively. The total sum of persipeptides enhancements resulted from the replacement of fermentation broth and subsequent optimization of their extraction by LLE reached almost seven-time, compared to conventional method (36.31 ± 1.37 μg mL-1). Therefore, relatively large amounts of persipeptides can be economically produced and extracted for various future experiments.