Mycoplasmas are resistance many types of antibiotics, it is very difficult to fight infection resulting in high morbidity. Nanoparticles are a viable alternative to antibiotics and appear to have high potential to solve the problem of bacterial drug resistance. The current study evaluated antimicrobial activity of tilmicosin with carbon nanoparticles on Mycoplasma bovigenitalium in vitro and in vivo laboratory rats. Twenty male albino Wistar rats with average body weight 100 g were used, divided into four groups (five rats per group). Group 1 was control negative. Group 2 was infected by intraperitoneal injection of M.bovigenitalium at a dose of 10⁵ CFU/mL. Group 3 was infected by M. bovigenitalium (10⁵ CFU/mL) and treated with tilmicosin only (0.5 mg/body weight). Group 4 infected by M. bovigenitalium and treated with tilmicosin + Carbon nanoparticle (0.35μg/ml). Tissue samples of testis were collected and fixed in 10 % neutral formalin buffer for histopathology. In Group 1 the testis exhibited normal histological structure. In Group 2 the testis demonstrated massive neutrophilic infiltration in the seminepherous tubules and interstitial tissue and complete necrosis of other tubules. Furthermore, there were areas in seminepherous tubules showed germ cell degeneration and multinucleated giant cell formation. Germ cell necrosis, multinucleated giant cell formation in seminepherous tubules and thickening of interstitial tissue with edema and leukocytes infiltration were also recorded. Bacterial aggregation was observed in the interstitial tissue. In Group 3 there were edema, multinucleated giant cell formation in seminepherous tubules and thickening of interstitial tissue with edema and leukocytes infiltration. Group 4, the seminiferous tubules were lined by spermatogenic cells to sperm formation. Advanced research must be done on antimicrobial nanoparticles will help in control of Mycoplasma infection in bovine.