The rising number of antibiotic-resistant bacterial strains represents an emerging health problem that has motivated efforts to develop new antibacterial agents. Endogenous cationic antibacterial peptides (CAPs) that are produced in tissues exposed to the external environment are one model for the design of novel antibacterial compounds. Here, we report evidence that disubstituted dexamethasone-spermine (D2S), a cationic corticosteroid derivative initially identified as a by-product of synthesis of dexamethasone-spermine (DS) for the purpose of improving cellular gene delivery, functions as an antibacterial peptide-mimicking molecule. This moiety exhibits bacterial killing activity against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa present in cystic fibrosis (CF) sputa, and Pseudomonas aeruginosa biofilm. Although compromised in the presence of plasma, D2S antibacterial activity resists the proteolytic activity of pepsin and is maintained in ascites, cerebrospinal fluid, saliva, and bronchoalveolar lavage (BAL) fluid. D2S also enhances S. aureus susceptibility to antibiotics, such as amoxicillin (AMC), tetracycline (T), and amikacin (AN). Inhibition of interleukin-6 (IL-6) and IL-8 release from lipopolysaccharide (LPS)- or lipoteichoic acid (LTA)-treated neutrophils in the presence of D2S suggests that this molecule might also prevent systemic inflammation caused by bacterial wall products. D2S-mediated translocation of green fluorescent protein (GFP)-labeled glucocorticoid receptor (GR) in bovine aorta endothelial cells (BAECs) suggests that some of its anti-inflammatory activities involve engagement of glucocorticoid receptors. The combined antibacterial and anti-inflammatory activities of D2S suggest its potential as an alternative to natural CAPs in the prevention and treatment of some bacterial infections.