Foodborne disease is a major public health problem. The present study examined Annona squamosa leaves, which are traditionally used to treat diarrhea and other infections, for their potential to be used in modern food safety or medicine. Active constituents were partially purified by ethanol extraction and column chromatography. MICs of the extract were 62.5 to 125 μg/mL against Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus, and 250 μg/mL against Campylobacter jejuni. In time-kill assays, 500 μg/mL of the extract reduced colony forming unit numbers of C. jejuni almost 10 000-fold within 12 hours. Similar decreases were seen against B. cereus, but over a longer time-frame. LC-MS analysis indicated the presence of reticuline and oxophoebine. Assessment of stability by MIC assay showed activity was heat-labile, with loss of activity greatest following high temperature treatments. Activity was relatively stable at refrigeration temperature. These results indicate A. squamosa has broad-spectrum but heat-labile activity against foodborne bacterial pathogens, and bactericidal activity against B. cereus and C. jejuni. This bactericidal activity is not sufficiently rapid for A. squamosa to be used as a food sanitizer, but the extract could potentially be developed as an additive for refrigerated foods, or a modern treatment for foodborne illness.