The aim of this research was to formulate, develop, and conduct an in vitro assessment of Neomycin nanoemulsion as an antimicrobial agent. Two formulations using Carbopol 934 and Carbopol 940 polymers were prepared and evaluated. The pH values for the two formulations were found to be compatible with skin pH, with mean pH values of 6.8 ± 0.05 and 7.1 ± 0.10 for Carbopol 934 and 940, respectively. The viscosity and spreadability of both formulations were measured, with the mean viscosity values of 51 ± 1.00 cP and 46 ± 1.41 cP, and mean spreadability of 8.5 ± 0.20 cm and 9.0 ± 0.10 cm for Carbopol 934 and 940, respectively. The mean particle size was within the nanometer range, with 122 ± 2.08 nm for Carbopol 934 and 130 ± 2.08 nm for Carbopol 940. The zeta potential values for both formulations were negative, indicating stability. In vitro drug release studies revealed a sustained release profile for both formulations. Overall, this research provides valuable insights into the development of effective antimicrobial nanoemulsions, showcasing their potential in the field of topical antimicrobial therapy