ABSTRACT Background: Iron deficiency anemia (IDA) has been the main cause of the health burden in India over the past decade. The aim of the present study was to examine the iron status and prevalence of iron deficiency anemia using biochemical markers among sanitation workers working at a tertiary healthcare center. Methodology: This hospital-based cross-sectional observational study was conducted on 80 sanitation workers for a period of 18 months (age 31–40 years). Dietary intake patterns were collected through the structured questionnaires. Blood samples were analyzed for complete blood count, serum iron level and unsaturated iron binding capacity, and ferritin. Total iron binding capacity and percentage transferrin saturation were calculated. Stool samples were screened for intestinal helminthic ova and cysts of intestinal protozoa. Results: Of the 80 sanitation workers, IDA was found in 38.7%; out of this, mild anemia was found in 16 (20%) participants, while 11.3% and 7.5% participants had moderate and severe anemia, respectively. Higher plasma sTfR levels were observed in women with severe iron depletion than in those with iron sufficiency (median 17.6 versus 15.5 nmol/L; P = 0.001). A statistically significant positive correlation between hemoglobin and serum iron, serum ferritin, and transferrin saturation was found. Hemoglobin was positively correlated with white blood cells, red blood cells, hematocrit, and mean corpuscular hemoglobin. Conclusion: This study highlights the presence of mild IDA among sanitary workers and lower levels of iron intake than the recommended dietary allowance.