Objectives Regular tooth brushing, at least twice a day, is effective for the control and prevention of dental caries and periodontal disease, and regular hand washing with soap can effective prevent infections. The study aimed to assess the prevalence, associated factors of oral, hand hygiene behaviour, and associated factors among adolescents in six Southeast Asian countries. Methods The sample consisted of 36,173 adolescent school children (median age 14 years), from Bangladesh, Indonesia, Laos, Philippines, Thailand, and Timor-Leste that participated in the cross-sectional “Global School-based Student Health Survey” (GSHS) in 2014–2015. Results Results indicated that 17.1% of students reported <twice a day tooth brushing and sub-optimal (not always) hand washing behaviour (44.8% before meals, 31.9% after toilet use, and 55.8% with soap). In adjusted logistic regression analysis, male sex, health risk behaviour (<daily fruit and <daily vegetable consumption, sedentary behaviour), psychological distress, and lack of peer and parental or guardian support were associated with sub-optimal oral hygiene, and male sex, health risk behaviour (tobacco use, < daily fruit, <daily vegetable consumption, sedentary behaviour, physical inactivity), psychological distress and lack of peer and parental support were associated with sub-optimal hand washing hygiene behaviour. Conclusions Poor oral and hand hygiene behaviour was identified. Several factors associated with poor hygiene behaviour were identified that can help in designing programmes for promotion of adequate oral and hand hygiene practices.