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• Inappropriate health care management system and poor implementation on standard in relevance to TB, remain some reasons causative to TB prevalence in high burden countries. • KAP study is important in order to optimize difficulties, limitations and capacities for enhancement of certain scenarios including health care. • We valuated a TB KAP study among Health Care Workers in ICT, Pakistan to highlight the KAP impact on TB prevalence in community. • For a better management system of infectious diseases like TB, a well-trained and professionally competent staff of Health Care Workers is far crucial. Tuberculosis (TB) as a foremost infectious disease adds massive burden to morbidity and mortality rate, despite of well-structured TB control programs around the globe. Inappropriate health care management system and poor implementation on standard in relevance to TB, remain some reasons causative to TB prevalence and its rising antimicrobial resistance. Health Care Workers (HCWs) laboring as a part of TB control system, are the vital warriors in achieving the goals of TB End Strategy by 2035. Their performance is influenced by their knowledge, attitude, and practices (KAP) toward this infectious disease. This study aimed to signify the role of KAP score of health care Workers in the better control and prevention of TB in the Islamabad Capital Territory (ICT), Pakistan. A cross-sectional study on Knowledge, Attitude and Practice study of Tuberculosis (TB) among health care Workers, was done in ICT, which is the capital of Pakistan. The KAP of TB was collected for the 306 Health Care Workers from all the Islamabad TB referring health facilities which refer the TB patients for testing to the National Reference Laboratory, Islamabad Pakistan. Eligible health care workers were requested to respond on KAP questionnaire after informed consent. KAP questionnaire comprised of knowledge, attitude, and practices section including demographic information. All the data was analyzed using IBM SPSS statistics 21. One Way Analysis of Variance (ANOVA) was applied to calculate KAP mean score against different variables. On the significant data sets of ANOVA output, Tukey's Multiple Comparison Test was applied for pairwise comparison. Pearson correlation coefficient was utilized to explore the association between two qualitative variables. The non-parametric tests were applied to evaluate difference of KAP score in relation to demographic covariates individually. From June to July 2023, we conducted TB KAP study among Health Care Workers of ICT, Pakistan. The average age was 33 years (range 26–30 years). Majority of the recruited subjects were not being trained for dealing with TB infection. The results demonstrated that Health Care Workers working were lacking their knowledge about mode of TB transmission, best diagnostic technique, and contraction of TB infections. The mean knowledge, attitude and practices mean scores were 15.05 (SD = 3.96), 83.68 (SD = 15.74) and 6.31 (SD = 2.21), respectively. Mean knowledge score of Health Care Workers were significantly related to their educational level and occupation while no significant association was declared with working experience as TB staff. Pearson coefficient of attitude score with knowledge of Health Care Workers was of weak level (0.28). Practice mean score was correlated to knowledge mean score at a moderate level (r = 0.40). On the other hand, practice score was r = 0.29 with attitude mean score had shown weak level correlation. A number of demographic factors were strongly linked to each of the mean score of knowledge, attitude, and practices. These findings highlighted the significant involvement of education, profession, and professional trainings in the better knowledge, attitude, and practices of the TB related health care Workers. For a better management system of infectious diseases like TB, a well-trained and professionally competent staff of Health Care Workers is important so as to achieve the goal of TB-End strategy by 2035 from Pakistan, which is the 5th highest burden country for TB. [ABSTRACT FROM AUTHOR]