The aim of this study was to evaluate the effects of preparation method and the type of surfactant on the properties of cephalexin (CPX) microspheres in order to obtain delivery systems suitable for the treatment of dairy mastitis. Microspheres were obtained using various preparation conditions and their physicochemical characteristics such as size, loading efficiency, morphology, and drug crystallinity were investigated. Antibacterial activity of microspheres from the optimum preparation condition was also studied. CPX microspheres were prepared by two different W/O/W emulsion solvent evaporation methods using PLGA as a matrix forming polymer. Several types of surfactants including nonionic, cationic, and anionic at different concentrations were used for preparation of the particles. The type and concentration of surfactant did neither affect the size nor morphology of the microspheres but showed a pronounced effect on the CPX encapsulation efficiency. It was found that Tween 80 showed the highest drug encapsulation efficiency (66.5%). Results from X-ray diffraction diffractograms and differential scanning calorimetry thermograms indicated that CPX entrapped in these microparticles was amorphous. Assessment of antibacterial activity showed that the obtained CPX microspheres exhibited good inhibition with minimum inhibitory concentration and minimum bactericidal concentration values of 128 microg/mL and 2,048 mg/mL against Staphylococcus aureus ATCC 25923, 512 microg/mL and 4,096 mg/mL against Escherichia coli ATCC 25922, respectively.