<p>The aim of the study was to evaluate the effects of Fluorothiazinone (FT), the inhibitor of type III secretion system (T3SS), on a model of a long-term infection in animal caused via the clinical isolate of <i>Burkholderia cenocepacia</i> acquired from the chronically infected cystic fibrosis patient. To simulate acute and long-term lung infection, DBA/2 mice were infected intranasally, the bacterial recovery from the lungs was assessed, and a histopathological study was performed. Intracellular survival and reproduction of bacteria in macrophages were evaluated on a monolayer of RAW264.7 cells. When simulating an acute infection, the isolate caused death of 50% of mice within 5 days, while combined preventive–therapeutic scheme with FT increased the survival rate to 100%. When simulating a prolonged infection, combined preventive–therapeutic scheme with FT resulted in decrease in lung colonization compared to the control group by several orders of magnitude on the 5th day after the infection and to complete eradication on the 10th day. Histopathological examination confirmed the efficacy of FT in reducing the intensity of lung damage. FT prevented survival of the isolate in RAW264.7 macrophages and reduced the ability of bacteria to replicate intracellularly. Thus the efficacy of the new non-traditional antibacterial agent FT has been shown in models of acute and in prevention of a long-term lung infections caused via a multidrug-resistant isolate <i>B. cenocepacia</i>. FT has reduced the ability of the isolate to survive intracellularly and replicate in macrophages, thereby affecting significant mechanisms of the development of chronic infections.</p>