Acinetobacter baumannii is one of the most challenging bacterial pathogens and poses a significant threat to modern medicine. The global significance of this Gram-negative, hospital acquired pathogen is primarily a result of its ability to rapidly develop drug resistance in clinical settings. Despite playing a major role as a barrier against antibiotic assault, our understanding of how composition of the bacterial membrane lipid bilayer influences drug resistance remains limited. This study explores the antimicrobial potential of the host-derived polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA), and its impact on antibiotic resistance. Assessment of antibiotic efficacy highlighted synergy of DHA with aminoglycoside antibiotics and pentamidine, which was subsequently attributed to DHA-induced conformational changes upon a primary multidrug efflux pump, AdeABC. In addition, DHA co-supplementation also reduced the gain of multidrug resistance, by interfering with efflux pump regulation. Overall, these analyses highlight the impact of DHA on antibiotic resistance and membrane protein behavior of A. baumannii. Further, this work provides novel insights into the potential utilization of DHA as an alternative treatment strategy against this superbug, either as direct antimicrobial agents or in combination with antibiotics to prevent the development of clinical drug resistance.