Herein, new microspheres were successfully fabricated from pentaerythritol tetrakis(3-mercaptopropionate) as model monomer and hexachlorocyclotriphosphazene as crosslinker using a single-step via precipitation polymerization method. Trimethoprim was chosen as the model antibiotic drug for drug release and antimicrobial activity studies. The percent of drug loading and drug entrapment efficiency of the microspheres was calculated to be 0.85% and 42.2%. According to correlation coefficients, the release kinetic was found to be Higuchi kinetic model and the release mechanism was non-Fickian model for microspheres. The drug-loaded microspheres exhibited excellent bacterial inhibition against gram-positive (Staphylococcus aureusandBacillus subtilis) and gram-negative (Escherichia coli) bacteria.