Microemulsions (μEs) are unique systems that offer exciting perspectives in biophysical research for mimicing biomembranes at the molecular level. In the present study, biocompatible μE formulation of a new oil-in-water (o/w) system comprising clove oil/Tween 20/2-propanol/water was accomplished for encapsulating an antibiotic, levofloxacin (LVF). The pseudoternary phase diagram was delineated at a constant cosurfactant/surfactant (2:1) ratio to meet the economic feasibility. The gradual changes occurring in the microstructure of the as-formulated four-component μEs were explored via multiple complementary characterization techniques. The results of electrical conductivity (σ), viscosity (η), and optical microscopic measurements suggested the existence of a percolation transition to a bicontinuous structure in the microregions of the as-formulated μE. LVF displayed a high solubility (5.0 wt %) at the pH of 6.9 in an optimum μE formulation comprising 2-propanol (36.4%), Tween 20 (18.2%), clove oil (20.7%), and water (24.7%). The LVF-loaded μE composition showed long-term stability for over 6 months of storage. Fourier transform IR analysis showed that LVF was stable inside the μE formulation, indicating the absence of any possible aggregation of LVF. Dynamic light scattering revealed that the average particle size of drug-free μE (64.5 ± 3.4 nm) increases to 129.7 ± 5.8 nm upon loading of LVF, suggesting the accumulation of LVF in the interfacial layers of the micelles. Moreover, fluorescence measurements indicated that LVF might be localized in the interfacial film of μE system, which may result in a controlled release of drug.