Topical drug delivery system is one that is applied directly to an external body surface either by innucting, by spraying or by dusting on or by instilling. The aim of this present research work was to get local action and reduces the side effects in contrast to oral dosage form. Trimethoprim is antibacterial drug and has been used in the treatment of bacterial infection such as boil or folliculitus caused by Staphylococcus aureus. Concentration of drug and Carbopol 940P were selected i.e. 1% & 2% respectively. Various penetration enhancers such as Six different penetration enhancers were used from different categories i.e. natural (Neem Oil and Menthol), semisynthetic (Ethanol and Propylene glycol), synthetic (Oleic acid and Caprylic acid). Three concentrations (2%, 5%, 10%) of each penetration enhancer were used to formulate F1 to F18 batches by using dispersion method . The variation in their concentration was studied for their effect on the drug release profile and permeation enhancement. All the formulation were investigated for homogeneity, pH, drug content, spreadibility, extrudability, rheological study, gel strength, in vitro diffusion study, in vitro microbial study and stability studies. Maximum cumulative % release obtained from the formulation F3, F9, F15 was 96.365, 80.949, 91.106 respectively. Formulation F3 having 10% Neem Oil was found to be the best formulation with maximum release of 96.365 and good inhibition zone.