To date, very little is known about avian mycoplasma infections in Tunisia. Mycoplasma gallisepticum is one of the most economically significant pathogens for poultry in Tunisia and worldwide. Based on the paucity of data regarding the genetic profiles and antibacterial behavior of M. gallisepticum strains in Tunisia, the present study was conducted. Genetic typing and phylogenetic relationships of 40 M. gallisepticum strains (20 Tunisian isolates, 19 international strains collection, and the S6 reference strain) were investigated by gene-targeted sequencing (GTS) using four loci (pvpA, mgc2, vlhA, and the intergenic spacer region between the 16S and the 23S rRNA genes). GTS revealed 12 sequence types (ST) that were found to spread over two clonal complexes (CC) and five singletons. Emergence of enrofloxacin and spiramycin resistance among M. gallisepticum local isolates has been revealed using the broth microdilution method. Causal mutations have been identified by sequencing the quinolone-resistance determining region and domain II and V of 23S rRNA as well as the rplD and rplV genes for enrofloxacin- and macrolide-resistant isolates, respectively. The emergence of antibiotic resistance to enrofloxacin and spiramycin has been identified as being related to a distinctive clonal complex formed by four different STs (ST2, ST3, ST4, and ST5), which would suggest that this phenotype was clonally disseminated.