Acinetobacter baumannii is one of the most problematic pathogens to treat due to the rapid development of resistance and long-term persistence in healthcare environments. Moreover, pandrug-resistant isolates have been increasingly reported worldwide. The development of novel antimicrobial drugs is needed, and microbial natural products are a promising source of bioactive compounds. This study proposed to evaluate the inhibitory potential of fluopsin C (FlpC), produced by the bioprospected Pseudomonas aeruginosa LV strain, on six extensively drug-resistant A. baumannii (XDRAB). The XDRAB isolates were recovered from tracheal secretion of inpatients of a public southern Brazilian tertiary hospital in Londrina (Paraná, Brazil) in 2019, and their susceptibility profiles were determined by automatized systems. The reference strain ATCC 19606 was also used. FlpC production by LV strain was performed in cupric nutrient broth (g/L: peptone 2; beef extract 1.2; CuCl2∙2H2O 0.03; pH = 6.8), inoculated with 108 CFU/mL of LV suspensions. The bioprocess was conducted for 8 d (28 °C and 170 rpm). Subsequently, cultures were acidified to pH 4, centrifugated (9000 rpm at 4 °C for 15 min), dried, and extracted with dichloromethane (2:1) three times. FlpC was purified by two sequential flash chromatography processes, using silica gel 60 as stationary phase. Mobile phases were composed of (I) dichloromethane:ethyl acetate (95:5) and (II) dichloromethane:ethyl ether (90:10). The antimicrobial activity of FlpC was determined by disk diffusion and broth microdilution methods, according to CLSI. For disk diffusion, disks were impregnated with 10 μg of FlpC and 10 μL of DMSO (negative control). The inhibition zones (mm) were measured after 24 h/37 °C incubation. In broth microdilution method, FlpC test concentrations ranged from 56 - 0.11 μg/mL. The plates were incubated for 20 h/37 °C. Disk diffusion assays resulted in inhibition zones ranging from 22.3 ± 0.58 mm (ATCC 19606) to 16.5 ± 0.71 mm (CI 226). The higher inhibition zone among XDRAB was 20.0 ± 1.41 mm (CI 232). The minimum inhibitory concentration (MIC) of FlpC for all XDRAB was equal to 3.5 μg/mL, whilst for ATCC 19606 was 1.75 μg/mL. FlpC demonstrated antibacterial activity against six XDRAB clinical isolates, and facing a lack of resources to combat these pathogens, within this specific resistance profile, we believe that FlpC is a promising microbial bioactive compound to drug development.