BackgroundTuberculosis (TB) remains a significant public health concern globally, particularly in regions with a high prevalence of HIV/AIDS. In Tanzania, the co-occurrence of TB and HIV poses a substantial challenge to healthcare systems. Isoniazid Preventive Therapy (IPT) has become an important intervention to mitigate the risk of TB infection among individuals living with HIV. Despite its proven efficacy, the extent of knowledge, attitude, and practice of healthcare workers (HCWs) in Tanzania is not well-documented. This study aimed to evaluate the levels of knowledge, attitude, and practice regarding IPT among healthcare workers.MethodsA cross-sectional study was carried out among healthcare workers working at TB/HIV clinics in 12 regions of Tanzania mainland. A semi-structured questionnaire was administered to the respondents consisting of social demographic characteristics, knowledge, attitude, and practice questions. Data were analyzed using Stata version 14.0.ResultsA total of 464 respondents were interviewed, with 282 (60.8%) being female. The mean age was 36.4 years (SD = 11.0). Among the interviewed, 255 (55.0%) were nurses. Regarding knowledge, attitude, and practice on IPT, 217 (46.8%), 220 (47.0%), and 422 (90.9%) respondents respectively demonstrated adequacy in these domains. Factors such as gender, university education, professional role in healthcare practice, cumulative work experience of one year or more, tenure at a TB/HIV clinic of one year or longer, and attendance at IPT-related training emerged as significant determinants. These factors influence respondents' knowledge, attitude, and practice towards IPT.ConclusionThe findings underscored a notable knowledge gap and a less favourable attitude towards IPT among HCWs, despite commendable practices. Addressing this disparity through targeted training interventions for healthcare workers holds promise for enhancing their understanding and attitude toward IPT, thereby fostering increased acceptance and utilization of this preventive therapy.