Background: Ineffective hand hygiene in healthcare settings is a global challenge that is associated with a high rate of nosocomial infections. The study aimed to measure the effectiveness of handwashing at Dilla University referral hospital. Method: This study consisted of 2 parts; the survey work and laboratory analysis. A total of 63 participants were selected to take surveys using an interviewer-administered questionnaire to collect the data regarding the socio-demographic and hand hygiene-related practices. A laboratory tests (swab test) was used to assess handwashing effectiveness from 63 participants by taking 126 swab test (63 before and after hand washing sessions). A swab test was collected from the palms of each participant before and after hand washing using a sterile technique. The cultures were then incubated aerobically overnight at 37°C, and examined for microbial growth. The bacterial load was reported as the number of colony-forming units (CFU). Result: The proportion of effective hand washing in Dilla University Referral Hospital was 82.5%. The mean colony-forming unit before and after handwashing were 55 and 2 CFU/ml, respectively with an average reduction of 94.6% in terms of CFU/ml. The mean amount of water used for effective handwashing was 336.03 (±219.46) ml. There was a significant mean difference in the amount of water used and duration of hand rubbing between effective and non-effective handwashing among the participants ( P < 0.01). The bacterial load before and after handwashing indicated that there was a significant (53.3 mean CFU) reduction in bacterial load after handwashing practice which indicated that the handwashing intervention in the Referral hospital was effective ( P < 0.01). Conclusion: The proportion of effective Hand washing in Dilla University referral Hospital was 82.5% with a 94.6% reduction in terms of (CFU/ml). The amount of water use and the duration of hand rubbing showed a significant difference in the reduction of the microbial load.