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Background: Zinc deficiency is evident in chronic diseases, but little is known about its association with multi-drug resistant tuberculosis (MDRTB). We aimed to measure serum zinc level in MDRTB patients and explore its association with MDRTB compared to drug-sensitive tuberculosis (DSTB). Methods: We recruited 107 MDRTB and 87 DSTB patients from a tuberculosis referral hospital in Bangladesh. After overnight fasting, 5 ml venous blood was collected from each patient to measure serum zinc level through graphite furnace atomic absorption spectrophotometry method. Multivariate logistic regression was done to measure its association with MDRTB. Results: The mean age of all patients was 36 years, where 70% were male. About 27% MDRTB patients and 2.3% DSTB patients had low serum zinc level (P < 0.0001). An inverse correlation was observed between serum zinc level and duration of anti-TB therapy (r-value: −0.252, P < 0.01). Reduced serum zinc level (odds ratio, 0.957; 95% confidence interval 0.923–0.992) was found as a significant associating factor for MDRTB after adjusted with age, sex, occupation, residence, tobacco consumption, Bacillus Calmette–Guérin-vaccination, and duration of anti-TB therapy. Conclusion: A lower serum zinc level is significantly associated with MDRTB. The tuberculosis control program should address this in the MDRTB control strategy.