This study seeks to determine the plasmid DNA profile of multidrug resistant organisms isolated from neonatal blood culture. The study design was prospective and specimens were collected in a specified order from neonatal intensive care unit (NICU). Neonates that were both in-born and out-born were used in this study. Specimens were collected from three hundred babies out of four hundred babies that were admitted into the neonatal intensive care unit (NICU) of the Hospital. One milliliter of blood was aseptically inoculated into each blood culture bottle of brain heart infusion broth and thioglycollate broth, they were incubated aerobically at 37ºC for up to seven days, and examined daily for bacteria growth using turbidity, bubbles and gas productions as an indication of presence of bacteria. Samples were cultured on MacConkey, Chocolate and Blood agar. Clinical isolates were identified to species level using the protocol of Cowan and Steel method. Antibiotic Susceptibility Testing (AST) was done using dilution method described by Fleming. Mueller Hinton Broth (MHB) was used as the diluents. Clinical isolates were subjected to Plasmid DNA profiling and curing test was carried out on multidrug resistant using ZymoPURE Plasmid Miniprep. This was followed by a post Plasmid curing test using Sodium Deodecyl Sulphate (SDS). 0.8% Agarose gel electrophoresis was carried out to separate the Plasmid DNA using 5µl ethidium bromate dye. Bands were visualized using ultra violet (UV) illuminator. Isolates that were multi-resistant yielded one or more plasmids. Plasmids curing made Pseudomonas aeruginosa, Proteus mirabilis and Klebsiella pneumonia thatwereformerly resistant to become susceptible. These findings suggest that environmental factors and genetic make- up of bacteria are important determinants of organisms’ susceptibility pattern.