INTRODUCTION: Hand hygiene (HH), one of the most important preventive measures of Healthcare-associated infections (HCAIs), is often neglected by healthcare workers (HCWs) in low and middle-income countries (LMICs). PURPOSE: The purpose of the study is to assess the role of a multimodal intervention (MMI) for enhancing hand hygiene compliance (HHC) of HCWs in a resource-limited setting. METHODOLOGY: A pretest-posttest quasi-experimental study was conducted in five hospitals of Bangladesh where 984 HCWs (342 physicians and 642 nurses) were selected purposively. Using a structured checklist, a direct observational assessment was carried out on HCWs’ HHC both before and after the intervention. The MMI provided to HCWs comprised of: (i) system change, (ii) educational intervention, (iii) visual reminders, (iv) monitoring and performance feedback and (v) formation of infection control committees. RESULTS: Following intervention, overall HH compliance before and after patient contact significantly increased (p<0.00) to 50.1% and 57.2% respectively across all hospitals, professional categories and activities. Nurses were more compliant to HH than physicians (OR = 1.1, 95% CI: 1.0-1.3, P < 0.01) after patient contacts. However, both groups showed equal HHC (OR = 1, CI: 0.9-1.1, P = 0.72) before patient contacts. HCWs of private hospitals were 1.5 times more compliant to HHC than that of public hospitals. CONCLUSIONS: This study denotes that despite national policies on hand hygiene in Bangladesh, HCW’s compliance to HH is poor. Study findings also illustrate that a multimodal HH program resulted in significant improvement in HCWs’ HHC that deserves the potentials to assist the advancement of infection control practices targeting reduction of HCAIs.