Wound infections caused by multidrug-resistant bacteria pose a significant challenge globally in healthcare. Traditional wound dressings often lack efficacy against these resilient pathogens, necessitating the exploration of innovative approaches to combat infections and promote wound healing. This study was designed to investigate a novel wound dressing marine extract-infused electrospun cellulose acetate nanofibers (CANF) with particular emphasis on combating multidrug-resistant bacteria. Cellulose acetate solutions were combined with marine extract (Heteroxenia Fuscescens soft coral, H. Fu) before the creation of electrospun nanofibers. The antibacterial activities of H. Fu extract and the prepared nanofiber mats were explored against Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, and Staphylococcus epidermidis. Furthermore, the antibacterial properties of the crude H. Fu extract were directly assessed, showing potent activity against several pathogens. Results displayed that H. Fu extract has a robust antibacterial action against tested bacteria. Different concentrations of H. Fu (0.05 g, 0.1 g, 0.2 g, and 0.3 g) were added to CA solution, and named as H. Fu-1@CANF, H. Fu-2@CANF, H. Fu-3@CANF and H. Fu-4@CANF, respectively. The obtained results from SEM analysis demonstrated the smooth and uniform fibers. As observed, the addition of H. Fu with specific concentrations (0.05 g, 0.1 g, 0.2 g) has no significant impact on the smooth properties of the formed nanofibers. Increasing the concentration of H. Fu (0.3 g) leads to the formation of nanofibers coated with huge beads. FTIR affirmed the existence of H. Fu that blended with CANF. The hydrophilicity of the formed CANF was significantly enhanced thanks to the presence of H. Fu extract. The contact angle values of CANF, H. Fu-1@CANF, H. Fu-2@CANF, and H. Fu-3@CANF were 33.3°, 57.1°, 60.8° and 62.9°, respectively. The mechanical properties in terms of ultimate force (kg) recoded values were 0.89, 0.95, 1.2, 1.3 for CANF, H. Fu-1@CANF, H. Fu-2@CANF and H. Fu-3@CANF, respectively, those of CNF were markedly enhanced when loaded with H. Fu. This study evaluates the antibacterial efficacy of nanofiber mats loaded with H. Fu extract against common cutaneous bacterial pathogens. Four nanofiber samples were tested for their inhibitory impact on bacterial survival counts using the disc diffusion method. Important results from the study include the observation that H. Fu-3@CANF exhibited the largest zones of inhibition (ZOI) with diameters of 35 ± 0.25 mm for P. aeruginosa, 32 ± 0.65 mm for A. baumannii, 28 ± 0.14 mm for S. aureus, and 27.6 ± 0.48 mm for S. epidermidis. Additionally, the H. Fu-2@CANF sample demonstrated strong antibacterial activity, with ZOI diameters of 32 ± 0.56 mm, 29 ± 0.81 mm, 26 ± 0.27 mm, and 25.4 ± 0.28 mm for the respective bacteria. The Microtox® assay further evaluated the toxicity levels of the nanofiber mats, revealing that while all samples exhibited some toxicity, the H. Fu-3@CANF sample had the lowest toxicity profile. These findings highlight the potential of H. Fu-loaded nanofibers, particularly H. Fu-3@CANF, for broad-spectrum antibacterial applications. The study underscores the importance of incorporating bioactive compounds into nanofiber mats to enhance their antimicrobial properties, making them suitable candidates for medical and environmental applications requiring effective bacterial eradication.