Padma is one of the largest rivers of Bangladesh, which has huge potential for fishery resources. Microbial pollution status of this riverine ecosystem has not been evaluated yet. Therefore, the present study was conducted to find out the bacterial pollution in sediment of this river. Four distinctive study locations were evaluated for three respective seasons in 2016. Enumeration of total heterotrophic bacteria (THB), total coliform (TC), fecal coliform (FC) and Vibrio cholerae were done by standard plate count technique from surface layer of sediment samples. Results revealed that all the samples were contaminated with THB, TC, FC and Vibrio cholerae with a range of 20.60×106±1.41 cfu/g (Site-2 Padma Garden) to 3.46×106±0.47 cfu/g (Site-4 Talaimari), 140.00×104±22.63 cfu/g (Site-2 Padma Garden) to 2.88×104±0.97 cfu/g (Site-1 T-Dam), 5.98×103±0.36 cfu/g (Site-2 Padma Garden) to 1.04×103±0.08 (Site-4 Talaimari) and 3.85×104±0.26 cfu/g (Site-3 I-Dam) to 0.91×104±0.07 cfu/g (Site-4 Talaimari), respectively. The bacterial density data had seasonality showing very high density during summer and winter seasons and the lowest in monsoon season. During the study period, it was observed that Site-2 (Padma Garden) and Site-3 (I-Dam) was comparatively more polluted with bacteria then other two sites. As per the criteria of sediment quality evaluation, the sediment of Padma River can be categorized as moderate to moderately high polluted with total heterotrophic bacteria, while very little polluted with fecal coliform bacteria. Therefore, the present study suggests a regular monitoring and assessment for reducing the bacterial load into the Padma River ecosystem through appropriate measures.