Microbial safety in food products is not always adequately controlled. Chemical antimicrobials which are recognized as hazards to human health are gradually replaced by natural antimicrobial compounds. In the current study, the antimicrobial activity against some Gram-positive and Gram- negative bacteria by the methanolic extract from rambutan fruit peels was evaluated using both in vitro (medium) and in situ (food matrices i.e. raw chicken breast and pangasius fillet fish) methods. Methanolic rambutan peel extract (lyophilized to powder with total phenolic content of 310 ± 14.5 mg GAE/g) with geraniin, ellagic acid, rutin, quercetin, and corilagin as main phenolic compounds was a potent inhibitor towards E. coli, V. campbellii, V. parahaemolyticus, V. anguillarum, P. aeruginosa, S. enteritidis, St. aureus, L. monocytogenes, and C. albicans using in vitro tests. In in situ tests, the extract inhibited S. enteritidis in raw chicken breast during 14 days of cold storage at 4 °C. Even though food matrices partly protected bacteria, the extract showed a 1.5 log CFU/g reduction of V. parahaemolyticus in fish during 10 days of cold storage. These results provide useful information on the utilization of rambutan fruit peel as natural antimicrobial agent in food products.