Amikacin is a very effective aminoglycoside antibiotic but according to its high toxicity, the use of this antibiotic has been limited. The aim of this study was to formulate and characterize amikacin loaded PLGA nanoparticles. Nanoparticles were synthetized using a solid-in-oil-in-water emulsion technique with different ratio of PLGA 50:50 (Resomer 502H) to drug (100:3.5, 80:3.5 and 60:3.5), two different concentrations of stabilizer (pluronic F68) (0.5% or 1%) and varied g forces to recover the final products. The most efficient formulation based on drug loading (26.0 ± 1.3 μg/mg nanoparticle) and encapsulation efficiency (76.8 ± 3.8%) was the one obtained with 100:3.5 PLGA:drug and 0.5% luronic F68, recovered by 20,000 × g for 20 min. Drug release kinetic study indicated that about 50% of the encapsulated drug was released during the first hour of incubation in phospahte buffer, pH 7.4, 37 °C, 120 rpm. Using different cell viability/cytotoxicity assays, the optimized formulation showed no toxicity against RAW macrophages after 2 and 24 h of exposure. Furthermore, released drug was active and maintained its bactericidal activity against Pseudomonas aeruginosa in vitro. These results support the effective utilization of the PLGA nanoparticle formulation for amikacin in further in vivo studies.