Colistin, a well-known Ab that combats gram-negative bacteria, has its antimicrobial effect restricted due to its nephrotoxicity side effect. Capsicum essential oil offers antimicrobial properties, but its poor solubility in water limits its usage in food. Incorporating it into a nanoemulsion can protect and enhance its application. The aim of our study is to examine the enhanced antimicrobial effect of capsicum oil nanoemulsion when combined with colistin against gram-negative bacteria. We used the agar well diffusion method to examine the antimicrobial effects of nanoemulsion, colistin, and their combination against Escherichia coli (ATCC 10536) and Klebsiella pneumoniae (ATCC 10031). TEM was used to examine the ultrastructure of the treated bacteria. Capsicum oil nanoemulsion droplets ranged in size from 41 to 123 nm, with a Z-potential of -25.8 mV and a PDI of 0.573, showing stability during our in vitro study. The combination of nanoemulsion with colistin exhibited a remarkable antimicrobial effect against Klebsiella pneumoniae, with an inhibition zone of 22±1.58 mm, compared to colistin alone, which had an inhibition zone of 18.60±1.14 mm. However, there was no difference in the inhibition zones for E. coli between the combined treatment and colistin alone, both showing an inhibition zone of 27.4±0.89 mm. TEM of gram-negative bacteria treated with both nanoemulsion and colistin showed clear cellular lysis and nuclear necrosis. Our results confirm that co-administration of colistin with capsicum oil nanoemulsion enhances the antimicrobial effect of colistin more effectively than its use alone.