Horizontal Gene Transfer was long thought to be marginal in Mycoplasma a large group of wall-less bacteria often portrayed as minimal cells because of their reduced genomes (ca. 0.5 to 2.0 Mb) and their limited metabolic pathways. This view was recently challenged by the discovery of conjugative exchanges of large chromosomal fragments that equally affected all parts of the chromosome via an unconventional mechanism, so that the whole mycoplasma genome is potentially mobile. By combining next generation sequencing to classical mating and evolutionary experiments, the current study further explored the contribution and impact of this phenomenon on mycoplasma evolution and adaptation using the fluoroquinolone enrofloxacin (Enro), for selective pressure and the ruminant pathogen Mycoplasma agalactiae, as a model organism. For this purpose, we generated isogenic lineages that displayed different combination of spontaneous mutations in Enro target genes (gyrA, gyrB, parC and parE) in association to gradual level of resistance to Enro. We then tested whether these mutations can be acquired by a susceptible population via conjugative chromosomal transfer knowing that, in our model organism, the 4 target genes are scattered in three distinct and distant loci. Our data show that under antibiotic selective pressure, the time scale of the mutational pathway leading to high-level of Enro resistance can be readily compressed into a single conjugative step, in which several EnroR alleles were transferred from resistant to susceptible mycoplasma cells. In addition to acting as an accelerator for antimicrobial dissemination, mycoplasma chromosomal transfer reshuffled genomes beyond expectations and created a mosaic of resistant sub-populations with unpredicted and unrelated features. Our findings provide insights into the process that may drive evolution and adaptability of several pathogenic Mycoplasma spp. via an unconventional conjugative mechanism.