Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG) are two of the most serious infections in poultry, causing financial losses across the globe. Antibiotics treatment can lessen the clinical signs of MG and MS infection in birds and help restrict the spread of these infections. However, the rise of antibiotics multidrug resistance (MDR) among poultry diseases is still a problem for the world's health. Minimum inhibitory concentration (MIC) is crucial to resolving this issue since it helps ensure correct antibiotic use and fights the development of antibiotic resistance. This investigation aims to assess the in vitro and in vivo efficacy of several antibiotics (tilmicosin, tylosin, erythromycin, spiramycin, lincomycin, and doxycycline) against MG and MS field isolates, in addition to guava leaves extract (GLE) against MG. For in vivo study, 510, one day old chicks were randomly split into 17 groups the birds were experimentally infected at 3 days old and supplied with different treatments in drinking waters for 5 successive days at 18 days old. The results of in vitro study showed that tilmicosin was the most effective against MG isolates with MIC values ranging from 0.0078 to 0.0156 μg/ml and spiramycin considered effective against MS with MIC values ranging from 0.015625 to16μg/ml and MIC value of GLE was 0.25 μg/ml that was considered as moderate sensitive to MG infection. The in vivo study revealed that tilmicosin and spiramycin were able to significantly lower the clinical score, lesion score, and re-isolation rate of MG and MS, respectively in the treated birds at p-value < 0.05 in contrast with control positive infected untreated groups. Additionally, the treated groups with tilmicosin in MG infection and spiramycin in MS infection showed significantly higher mean body weights (MBW) compared to the infected untreated ones at p-value < 0.05. The current study demonstrates that MG and MS's sensitivity to many of the most popular drugs changed as in tylosin become low sensitive and erythromycin become resistant. GLE has moderate sensitivity antibacterial effect against MG isolates both in vivo and in vitro and can be used as a natural antibiotic alternative. Therefore, we recommend periodical monitoring of the sensitivity of the circulation MG and MS strains using MIC test to overcome MDR of avian mycoplasmas with further suggestion to evaluate GLE as nano-preparation against MG infection.