Healthcare associated infections (HAIs) are a burden in many countries especially low-income countries due to poor hand hygiene practices in the healthcare settings. Proper hand hygiene in the healthcare setting is an effective way of preventing and reducing HAIs, and is an integral component of infection prevention and control. The objective of this study was to determine adherence to hand hygiene guidelines and associated factors among nurses and clinicians. A quantitative cross-sectional study was conducted at Chiradzulu District Hospital (Malawi) where stratified random sampling was used to obtain the sample of 75 nurses and clinicians. Data were collected using self-administered questionnaires (n = 75), observation checklists (n = 7) and structured observations (n = 566). The study findings confirmed low adherence to hand hygiene practice among healthcare workers (HCWs) in Malawi. Overall, higher hand hygiene practices were reported than observed among nurses and clinicians in all the World Health Organization’s (WHO) five critical moments of hand hygiene. This calls on the need for a combination of infrastructure, consumables (e.g., soap) and theory driven behavior change interventions to influence adoption of the recommended hand hygiene behaviors. However, such interventions should not include demographic factors (i.e., age, profession and ward) as they have been proven not to influence hand hygiene performance.