Objectives Healthcare workers (HCWs) are at high risk of latent tuberculosis infection (LTBI) due to their continued occupational exposure. The incidence of LTBI among the HCWs has seldom been investigated. Primary health centres (PHCs) provide effective and affordable medical care largely for the local population. The HCWs of PHCs who are likely to have increased occupational exposure have an increased risk of reactive as well as LTBI, and contribute to overall TB transmission than the general population. Methods A cross-sectional study (March–April 2024) was carried out to assess the burden of LTBI among the HCWs of PHCs (n=64) across Chennai, India. Blood samples were analyzed for gamma interferon using a QuantiFERON–TB Gold Plus assay. A complete baseline health profile, including complete blood count, blood glucose, liver and renal functions, and ferritin levels was estimated to identify potential biomarkers that are independently associated with LTBI. Results The present study revealed an LTBI prevalence of 25.3% (n=99) among PHC workers. The red cell distribution width was significantly associated with LTBI positivity among different biochemical and haematological parameters analyzed. Factors such as individuals age, underlying comorbid conditions (30.3%; n=30), and longer employment duration (28%; n=28) were significantly associated with IGRA positivity. IGRA positivity was significantly associated with decreased RDW–SD in females, overweight, and participants with O blood group. Conclusions The study reported a high prevalence of LTBI among HCWs of PHCs, which necessitates their periodical screening for the elimination of TB.