S. Typhimurium biofilm formation on chicken and food-contact surfaces presents a major challenge to food safety by enhancing bacterial persistence and resistance to sanitation. This study investigated the efficacy and mechanisms of cinnamon leaf oil (CLO), a sustainable plant-based antimicrobial, in controlling S. Typhimurium (ATCC 14028) biofilm formation. CLO exhibited a minimum inhibitory concentration (MIC) of 0.09% (v/v) (equivalent to 0.9 μL/mL). At concentrations ranging from ¼× to 4× MIC, CLO significantly (p < 0.05) reduced biofilm formation on chicken meat and reduced bacterial populations within preformed biofilms on food-contact surfaces including low and high-density polyethylene, polyethylene terephthalate, rubber and stainless steel in a dose-dependent manner. Mechanistic analyses revealed that CLO delayed bacterial motility, reduced cell surface hydrophobicity by approximately 60% at 4× MIC and inhibited autoaggregation. Confocal laser scanning microscopy demonstrated concentration-dependent inhibition of biofilm formation, with biofilms formed in the presence of CLO appearing progressively thinner, less densely organized and containing a higher proportion of non-viable cells as the CLO concentration increased. Collectively, these findings indicate that CLO inhibits the early stages of biofilm formation by interfering with bacterial adhesion and colonization, while also reducing bacterial populations within pre-established biofilms on food-contact surfaces.