Healthcare workers (HCWs) are at increased risk of infection with Mycobacterium tuberculosis (Mtb) and, hence, of developing tuberculosis (TB) disease. The aims of this study are to identify the prevalence and determinants of latent TB infection (LTBI) among HCWs in Duhok Province. This is a cross-sectional prospective study conducted during April–July 2018 in different health care facilities of Duhok province. HCWs at multiple levels were selected by a non-systematic random sampling method. Information on demographic and associated risk factors of LTBI were collected by using a standardized questionnaire. Thereafter, all HCWs underwent QuantiFERON Gold Plus (QFT-Plus) assay. HCWs with indeterminate QFT-Plus underwent a Tuberculin Skin Test. HCWs with positive results were further evaluated by smear microscopy investigation and chest X-ray examination. Three hundred ninety-five HCWs were enrolled; 49 (12%) tested positive for LTBI. The mean age of the HCWs was 33.4 ± 9.25 with a female predominance (51.1%). According to the univariate analysis, LTBI was significantly higher among HCWs with the following: age groups ≥ 30 years, alcohol intake, ≥ 11 years of employment, high risk stratification workplaces, and medical doctors. In the multivariate analysis, the age group of 30–39 years (OR = 0.288, 95% CI: 0.105–0.794, p value = 0.016) was the only risk factor associated with LTBI. Further medical investigations did not reveal active TB cases among HCWs with LTBI. With regards to prophylactic treatment, 31 (63.3%) LTBI HCWs accepted the treatment, whereas 18 (36.7%) declined the chemoprophylaxis. Of these 31 HCWs on chemoprophylaxis, 12 (38.7%) received isoniazid (INH) for six months, 17 (54.8%) received INH in combination with rifampicin (RMP) for three months, and two (6.5%) received alternative therapy because of anti-TB drug intolerance. In conclusions, although Iraq is a relatively high TB burden country, the prevalence of LTBI among Duhok HCWs is relatively low. It is important to screen HCWs in Duhok for LTBI, particularly medical doctors, young adults, alcoholics, and those whom had a long duration of employment in high-risk workplaces. The acceptance rate of HCWs with LTBI to chemoprophylaxis was low. Therefore, ensuring medical efforts to educate the healthcare staff particularly, non-professionals are a priority to encourage chemoprophylaxis acceptance.