In this paper, rapid, easy and cheap sonochemical method was described for synthesis of florfenicol-chitosan nanocomposite and evaluating its anti-bacterial effect against Escherichia coli (ATCC35218), Salmonella typhymurium (ATCC14028) and Staph. aureus (ATCC29213). Florfenicol-chitosan nanocomposite was full characterized for index, identification and morphology properties. Characterization results showed zeta sizing of florfenicol was 30.6 nm, while florfenicol-chitosan nanocomposite was 67.22 nm with zeta potential -14.5 and -28, respectively. Brunner-Emmett-Teller theory (BET) surface area was found to be 13.3, 73.2 and 103.69 m2/g for florfenicol, chitosan nanoparticles and florfenicol-chitosan nanocomposite, respectively. Raman charts confirmed the formation of florfenicol-chitosan nanocomposite without any contamination. Transmission electron microscope (TEM), scanning electron microscope (SEM) and atomic force microscope (AFM) images and data illustrated spherical to sub spherical shape of florfenicol nanoparticles on sheet shape of chitosan with size less than 75 nm. Remarkable results of florfenicol-chitosan nanocomposite as anti-bacterial agent illustrated the power of nanotechnology. However, antimicrobial activity was screened where the zone of inhibitions caused by the prepared nanocomposite were 24.7 mm, 30.6 mm and 29.3 mm compared to 17.7 mm, 16 mm and 18.7 mm of the native drug against E. coli, Salmonella typhymurium and Staphylococcus aureus, respectively.