This cross-sectional study was carried out in BNS Patenga- a Naval Hospital, Chittagong from February to June 2017 to knowthe knowledge and compliance of hand decontamination practice among health care workers with a sample size of 150following purposive sampling technique using a pre-tested semi structured questionnaire by face-face interview method. Thestudy revealed that majority of the respondents 114(76%) were males. This study also found that most of the respondents hadeducation up to SSC level 59(39.3%), followed by Diploma/Graduate / Masters 39(25.5%) and HSC 32(22%). Mean age ofrespondents were 32.40 years with a SD of ±7.366. This study revealed that 40(27%) respondents were from age group 31-35years, 33(21.9%) were both from age group ≤ 25 years and 36-40 years, 27(18%) respondents were from 26-30 years age groupand 17(11.2%) were of > 40 years of age. Compliance of hand decontamination practice was significantly associated with sex,designation, education and work place (p<0.05). There was statistical significant association between lack of knowledge, timeconstraints, lack of facilities, irritation or dryness of hands, facilities are inconveniently located, beliefs that use of glovesobviates the use of hand hygiene) and compliance to hand washing (p<0.002); also there were significant association betweencompliance of hand decontamination and availability of soap, availability of alcohol mixed substance, and distance ofavailable facilities (p<0.001). To conclude, this study has clearly shown the requirement of an in-depth appraisal of educationalinterventions to recognize the hand hygiene opportunities and improved availability of hand hygiene facilities.