Mycobacterium tuberculosis and HIV constitute a public health challenge. Health workers (HWs) in HIV clinics maybe at greater risk of M. tuberculosis infection, considering the high rates of HIV/tuberculosis (TB) coinfection among patients. Hence, we measured the prevalence of M. tuberculosis infection and the effect of working in an HIV clinic. We conducted a cross-sectional study in high-HIV burden health-care facilities in Abuja and Nasarawa states and recruited HWs over 4 months. We administered questionnaires and screened for M. tuberculosis infection using QuantiFERON-TB Gold-Plus. A total of 1,043 HWs were enrolled, with the majority being clinical staff (77.4%). Prevalence of interferon gamma release assay (IGRA) positivity was 44.8% (43.8% among HWs from HIV clinic and 45.3% from non-HIV clinics, P = 0.24). Nonoccupational factors such as living in a moderately (odds ratio [OR] = 0.71] or sparsely populated neighborhood (OR = 0.56), remained associated with a reduced risk of IGRA positivity, whereas male gender (OR = 1.37) and having high blood pressure (HBP) (OR = 1.52) remained associated with an increased risk after adjusting. Occupational factors such as length of career as a HW of 10 to 20 years (OR = 1.45) or 20 to 30 years (OR = 1.74) remained associated with an increased risk of IGRA positivity after adjusting. In a final multivariate model, the factors of age between 20 to < 30 years (OR = 0.61), having HBP (OR = 1.56), having a length of career as a HW of 10 to 20 years (OR = 1.66) or 20 to 30 years (OR = 2.09) and being a clinical HW (OR = 0.62) remained associated with IGRA positivity. There is a high prevalence of IGRA positivity among HWs in Nigeria. Working in HIV clinics, however, is not associated with increased risk.