Introduction – This work proposed to use neem (Azadirachta indica) seed oil as an antimicrobial substance to improve the microbiological safety and quality of fresh-cut mango (Mangifera indica L.). Neem oil (NO) from seeds was tested in vitro and in vivo in volatile form or in incorporation into tryptic soy agar medium with nalidixic acid (TSAN) or in taro (Colocasia esculenta) starch based coating. Materials and methods – Antimicrobial effects of the volatile compounds of NO (at 30, 60, 80, 100 or 500 µL) and their incorporation (1% v/v) have been studied by two ways: in the medium of culture and in coating. Tests were done on the in vitro and in vivo growth of natural microbial flora, particularly the total aerobic mesophilic flora, total coliform, enterobacteria, yeasts, molds, and specifically on the inhibition of the growth of Escherichia coli K12 and Salmonella enterica subsp. enteritidis in fresh-cut mango. Results and discussion – The NO volatile were ineffective in vitro. However, the dose of 500 µL greatly reduced the growth of common flora of fresh-cut mango fruit, allowing considering an in vivo study of their antimicrobial activities. In vivo, the NO volatiles reduced the growth of both organisms at 8 and 10 days of storage at 4 °C. Neem oil incorporated in the culture medium slightly reduced the growth of E. coli K12 and had no significant effect on S. enterica subsp. enteritidis. Its incorporation into the starch coating was ineffective against the Salmonella strain and slightly reduced the growth of E. coli K12. Conclusion – This is the first study reporting the NO from seeds as potential antimicrobial substance against pathogenic and spoilage bacteria in fresh-cut fruit. Its use in food will require further studies to check the most effective dosage and perform the related toxicological tests.