Aim To isolate and characterize strains of Mycoplasma agalactiae from bulk tank and silo ewes' milk. Methods and Results Thirteen mycoplasma isolates were obtained from samples of sheep milk taken from bulk tank and large silos and identified as Myc.agalactiae by PCR-DGGE. The isolates were typed by pulsed field gel electrophoresis (PFGE), SDS-PAGE and immunoblot. The in vitro activity of 13 antimicrobials of veterinary interest was tested against these isolates. Results showed that the most effective compounds against Myc.agalactiae in vitro were clindamycin, an antibiotic not previously described as a suitable contagious agalactia (CA) treatment, with Minimum Inhibitory Concentration (MIC) values of <0 center dot 12gml1, and quinolones, with MIC values <0 center dot 120 center dot 5gml1, which are used as standard treatments against CA. Conclusions Based on the in vitro assay, clindamycin, quinolones, tylosin and tilmicosin would be appropriate antimicrobials for CA treatment. The isolates were mostly resistant to erythromycin, indicating that it would not be a suitable choice for therapy. The isolates showed common molecular and protein profiles by PFGE and SDS-PAGE, with minor differences observed by immunoblot analysis, suggesting a clonal relationship among them. Significance and impact of the Study This study demonstrated the importance of the appropriate selection of antimicrobials for treatment of CA.