BackgroundEffective hand hygiene is crucial for preventing healthcare-associated infections (HAIs) among healthcare workers (HCWs) and patients. This study aimed to assess handwashing practices among HCWs providing routine HIV care to People Living with HIV (PLHIV) and to compare hand hygiene patterns among clinical and non-clinical health workers in APIN Public Health Initiatives-supported states in Southwest Nigeria.MethodsA cross-sectional study was conducted among HCWs providing routine HIV care to PLHIV in selected healthcare facilities across APIN-supported states (Oyo, Ogun, and Ondo) from January to June 2024. Data on handwashing patterns and compliance factors were collected using a semi-structured questionnaire. A total of 588 respondents were recruited using multi-stage stratified cluster sampling. Descriptive analysis was performed, hand hygiene patterns among clinical and non-clinical health workers were compared, and chi-square statistics determined the associations, with a p-value of < 0.05 considered significant.ResultsThe study found that 56.5% of participants had good awareness of handwashing practices, and 54.6% demonstrated good compliance. Additionally, 65.5% reported washing hands before and after routine activities. Handwashing awareness (60.3%) and compliance (60.5%) rates were highest among clinical professionals. Major barriers to handwashing included inadequate water supply, lack of materials, being too busy, and forgetfulness. Profession was significantly associated with hand hygiene awareness (odds ratio [OR] = 1.504, confidence interval [CI] = 1.075–2.104), while age (OR = 0.613, CI = 0.442–0.851) and profession (OR = 1.888, CI = 1.347–2.645) were significantly associated with compliance.ConclusionThe study highlights suboptimal handwashing practices among healthcare workers, with non-clinical workers demonstrating lower awareness and compliance to hand hygiene standards compared to clinical staff despite working in the same environment. Therefore, future tailored training sessions and interventions should be prioritized for non-clinical health workers.