Lymph node enlargement is a common presenting complaint in outpatient and inpatient department. The present observational cross sectional study was conducted in department of Internal Medicine, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh during the period from December 2014 to May 2016 to evaluate etiologies of significant lymphadenopathy by clinical, histopathological and microbiological assessment. Biopsy/FNA materials of 177 patients of 18-75 years age range with significant lymphadenopathy were sent for histopathology/cytology, Gram stain & culture, AFB stain & culture and Gene Xpert. Among them, 102(57.62%) were granulomatous lymphadenitis, 52(29.38%) were lymphoma, 12(6.78%) reactive lymphadenitis, 7(3.95%) metastatic malignancy, 2(1.13%) atypical lymphoid hyperplasia, 1(0.57%) myeloid sarcoma and 1(0.57%) chronic sialadenitis. Growth of MTB was on 23(22.55%) cases; among 102 granulomatous lymphadenitis and Gene Xpert was positive in 73(71.56%) cases with 100% Rif. sensitive. Gene Xpert is an important tool for diagnosis of tuberculous lymphadenitis. Time of symptoms to diagnosis of most of the TBL patients was within 2-8 months.