In Mycoplasma agalactiae, two simultaneous processes of DNA transfer have been described that require direct cell-to-cell contact and are similar to conjugation. One involves the self-transmission of an integrative conjugative element (ICE) while the second concerns the horizontal transfer of large and small fragments of chromosomal DNA. Here, we describe an optimized conjugation protocol for the horizontal transfer of ICE or chromosomal DNA carrying antibiotic resistance markers (i.e., tetracycline, gentamicin, puromycin) from donor to recipient mycoplasma cells. Calculation of the conjugation frequencies, selection and characterization of transconjugants are detailed. This protocol has been developed with M. agalactiae but has been successfully used for M. bovis and can be adapted to other related mycoplasma species.