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Objectives To screen novel small molecule compounds for inhibition of Mycoplasma bovis growth and to characterize their activity in terms of dose-dependency and ability to function in milk. Methods Using a tetrazolium salt cytotoxicity assay, 480 natural compounds were screened to determine which of the small molecules have the potential to become therapeutic options for M. bovis prevention and treatment. The dose response was determined in broth culture and in fresh quarter milk for a subset of compounds shown to be capable of inhibiting M. bovis growth. Results Data suggest that 32 of the 480 compounds tested were able to inhibit growth of M. bovis using a tetrazolium salt assay. Methanesulphonic acid, 3-[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyloxy](1S,3R,4R,5R)-1,4,5-trihydroxycyclohexane carboxylic acid, S-carboxymethyl-l-cysteine, l-aspartic acid, dihydrotachysterol, eriodictyol and (+)-α-tocopherol acid succinate were selected for further concentration-dependent studies and testing in fresh quarter milk. Each compound demonstrated a dose response in broth culture and at 3 h and 24 h in fresh quarter milk. Conclusions Small molecule natural compounds are capable of inhibiting the growth of M. bovis in both a pleuropneumonia-like organism (PPLO) medium and in fresh quarter milk. Results suggest that the compounds are mycoplasmastatic in a dose-dependent manner. By inhibiting M. bovis, small molecule natural compounds offer the potential for prophylactic or therapeutic use on organic and natural farms as a viable alternative to traditional antimicrobial agents. [ABSTRACT FROM PUBLISHER]