Biofilms represent a major challenge in treating bacterial infections due to their enhanced resistance to antibiotics. While probiotic bacteria such as Lactobacillus plantarum are known for their health benefits and ability to form protective biofilms, the impact of antibiotics on their biofilm-forming capacity remains underexplored. This study investigated the effects of subinhibitory concentrations of four antibiotics: Gentamicin, Ciprofloxacin, Amoxicillin-Clavulanic acid, and Trimethoprim-Sulfamethoxazole (TSH), on L. plantarum biofilm formation. The minimal inhibitory concentrations (MICs) were determined, revealing Gentamicin as the most potent antibiotic against L. plantarum. Biofilm quantification demonstrated that Gentamicin significantly reduced biofilm formation at concentrations near its MIC, while TSH induced weak biofilm formation at higher sub-MIC levels. Ciprofloxacin and Amoxicillin-Clavulanic acid showed no notable impact on L. plantarum biofilm development. These findings underscore the complex interaction between antibiotics and probiotic biofilms, highlighting the importance of antibiotic selection to preserve probiotic functionality and gut microbiome stability. Furthermore, the study offers valuable insights for developing probiotic-enhanced biomaterials where controlled biofilm formation is crucial.