Introduction: Antimicrobial substances (AMS) produced by bacteria may reduce or prevent the growth of pathogenic and spoilage microorganisms in food. In this study, 16 isolates of Acinetobacter baumannii/calcoaceticus (ABC) complex, previously obtained from reconstituted infant milk formula (IMF) samples and the preparation and distribution utensils from the nursery of a public hospital, were used to screen for AMS production. Methodology: Antimicrobial substance production and spectrum of activity assays were performed by agar-spot assay. Optimization of growth conditions for AMS production was also evaluated. Results: Three (17.6%) isolates, namely JE3, JE4, and JE6, produced AMS against the principal indicator strain Salmonella enterica subsp. enterica serotype Typhi ATCC 19214. JE6 was also able to inhibit strains of Klebsiella pneumoniae, Proteus vulgaris, and Bacillus cereus, a Gram-positive bacteria. Remarkably, JE6 was able to inhibit all the tested resistant and multidrug-resistant (MDR) strains of the ABC complex and Shigella dysenteriae associated with IMF and utensils, indicating a potentially valuable application. AMS produced by JE6 does not appear to be affected by proteolytic enzymes and the producer strain showed specific immunity to its own AMS. Conclusion: This study highlights AMS produced by Acinetobacter with applications against MDR spoilage and foodborne pathogens - some of them, infectious disease causing agents - which, to our knowledge, has not been previously described.