A cross-sectional survey utilizing questionnaire assessed veterinarians’ awareness, attitudes and practices of hand hygiene (HH) in Enugu State Southeastern Nigeria. Data obtained were analyzed using chi-square on SPSS (version 15.0) at a significance level of P < 0.05 to determine possible associations between variables and perceptions/knowledge about HH. Out of 294 respondents, 26.9% were familiar with the WHO’s “5 moments of HH” model but majority knew the importance of the model. There was high compliance with HH after touching an animal (100%) and animal’s surrounding (96%), and contact with body fluid (100%) but HH before touching an animal (52.5%) and performing clean procedures (58.5%) were practiced sub-optimally. Majority of the respondents often forgets to perform HH (61.9%) and prioritizes clinical tasks to HH when busy (65%). The attitudes/practices towards HH were generally negative. Minority knew that unclean veterinary personnel’s hands are the main route of infection in the veterinary clinic (38.1%) and alcohol-based hand rubs are more effective on pathogens than other cleansing agents (23.1%). Majority of the respondents does not know when different HH techniques ought to be performed. The overall knowledge of HH by the respondents was moderate (55.4%). No significant association (P > 0.05) was found between level of knowledge and gender, age, qualification, duration of practice and location. Poor knowledge of HH guidelines, unavailability and inaccessibility of HH facilities, and unavailability of reminders were the major barriers to compliance with appropriate HH practices. In conclusion, veterinarians’ awareness and perception about HH in the study area is moderate while the practices are sub-optimal, thus the education of Nigerian veterinarians about HH and the teaching of the principles and practices of appropriate HH during veterinary schools are recommended.