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The present study was conducted to investigate the internal and external biosecurity levels and their association with Mycoplasma gallisepticum infection in broiler farms in Khartoum State, Sudan. Sixty-six broiler farms were selected. A questionnaire of 77 questions on internal and external biosecurity measures was administered during each farm visit. A total of 360 blood samples were collected and used for serological examination to detect antibodies against M. gallisepticum. The overall score of biosecurity was 0.57, and the average score of internal biosecurity (0.75) was higher (p<0.01) than the average score of external biosecurity (0.51). Poor internal biosecurity practices were observed in 23.4% of broiler farms, whereas most farms (93.6%) recorded poor external biosecurity. A significant (p<0.01) positive correlation was encountered between the external biosecurity and the number of houses (0.62), as well as the number of workers (rs= 0.54). Both internal and external biosecurity displayed significant positive correlations with the number of birds (rs= 0.64, 0.35; p< 0.05). Fifty percent of the farms were positive for M. gallisepticum. The disease incidence correlated positively with the spread of manure on neighboring farms (rs= 0.44, p<0.05). A negative correlation was observed between the presence of Mycoplasma and the distance between farms (rs= -0.50, p<0.01) as well as farm enclosure (rs= -0.44, p<0.05). It is concluded that broiler farms in Khartoum State exhibited low biosecurity measures and a high seroprevalence of M. gallisepticum infection. Further efforts are necessary to improve external biosecurity practices. [ABSTRACT FROM AUTHOR]