Biofilms formed by foodborne pathogens represent a significant threat to food safety and public health. Natural plant-derived oils, such as Passiflora seed oil, may offer an effective alternative to conventional antibiofilm agents. The antibiofilm activity of Passiflora seed oil (10 and 20 μg/mL) was evaluated against five relevant pathogens (A. baumannii, E. coli, L. monocytogenes, P. aeruginosa, and S. aureus) using crystal violet (CV) as well as its capacity to affect the metabolism of microbial sessile cells (using the MTT test). Both assays were conducted at time zero (CV0, MTT0) and after 24 hours of bacterial growth (CV24 and MTT24) to evaluate their efficacy in inhibiting biofilm formation and disrupting mature biofilms. At 20 µg/mL, the oil strongly inhibited biofilm formation (CV0) caused by A. baumannii (61.2%), E. coli (44.7%), and L. monocytogenes (72.5%). However, activity against pre-formed biofilms (CV24) was limited except for S. aureus (64.6%), E. coli (19.31%), and L. monocytogenes, against which the oil exhibited very weak inhibitory activity (4.85%). MTT0 results only showed a significant reduction in metabolic activity of sessile cells for E. coli (84.4%); on the contrary, the effect of the oil was seen against all the strains when it was added after 24 h (MTT24), and the sessile cells of P.aeruginosa, the biofilm of which was completely resistant to the oil, were very sensitive to the presence of the oil (73.01% of inhibition at MTT24). Passiflora seed oil exhibits strain-specific and time-dependent antibiofilm properties, with strong preventive effects and selective action on mature biofilms. These findings support its potential as a natural antimicrobial for food safety applications.