Objectives: Serratia marcescens is a gram-negative pathogen of many species. The ability of S. marcescens to form biofilms and its potent innate resistance to antimicrobials and cleaning solutions are both essential for its pathogenicity and survival. The present study was conducted to investigate the effect of glyceryl trinitrate (GTN) on the biofilm of S. marcescens, as an alternative for antibiotic therapy. Methods: Different specimens, including ear swabs, burns, mid-stream urine, wound swabs, and sputum, were collected from patients who were brought to Al-Ramadi Hospital, Iraq. All samples were cultured, and the colonies that were obtained were detected using the VITEK® 2 compact. The ability of biofilms to develop was examined using the microtiter plate technique. The bactericidal effectiveness of GTN was estimated by the broth microdilution technique. The presence of fimA and fimC in S. marcescens isolates was detected using the polymerase chain reaction (PCR) method. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess the effect of GTN on fimA and fimC gene expression. Results: The results demonstrated that GTN has no effect on S. marcescens growth; while its biofilm was significantly (p<0.05) influenced. Moreover, all S. marcescens isolates had fimA and fimC, and the presence of GTN reduced the expression of these genes. Conclusion: The findings of this study reveal that GTN can act as a promising antibiofilm agent in reference to S. marcescens.