Chatbots (also known as conversational agents and virtual assistants) offer the potential to deliver healthcare in an efficient, appealing and personalised manner. The purpose of this systematic review and meta-analysis was to evaluate the efficacy of chatbot interventions designed to improve physical activity, diet and sleep. Electronic databases were searched for randomised and non-randomised controlled trials, and pre-post trials that evaluated chatbot interventions targeting physical activity, diet and/or sleep, published before 1 September 2022. Outcomes were total physical activity, steps, moderate-to-vigorous physical activity (MVPA), fruit and vegetable consumption, sleep quality and sleep duration. Standardised mean differences (SMD) were calculated to compare intervention effects. Subgroup analyses were conducted to assess chatbot type, intervention type, duration, output and use of artificial intelligence. Risk of bias was assessed using the Effective Public Health Practice Project Quality Assessment tool. Nineteen trials were included. Sample sizes ranged between 25–958, and mean participant age ranged between 9–71 years. Most interventions (n= 15, 79%) targeted physical activity, and most trials had a low-quality rating (n= 14, 74%). Meta-analysis results showed significant effects (allp< 0.05) of chatbots for increasing total physical activity (SMD = 0.28 [95% CI = 0.16, 0.40]), daily steps (SMD = 0.28 [95% CI = 0.17, 0.39]), MVPA (SMD = 0.53 [95% CI = 0.24, 0.83]), fruit and vegetable consumption (SMD = 0.59 [95% CI = 0.25, 0.93]), sleep duration (SMD = 0.44 [95% CI = 0.32, 0.55]) and sleep quality (SMD = 0.50 [95% CI = 0.09, 0.90]). Subgroup analyses showed that text-based, and artificial intelligence chatbots were more efficacious than speech/voice chatbots for fruit and vegetable consumption, and multicomponent interventions were more efficacious than chatbot-only interventions for sleep duration and sleep quality (allp< 0.05). Findings from this systematic review and meta-analysis indicate that chatbot interventions are efficacious for increasing physical activity, fruit and vegetable consumption, sleep duration and sleep quality. Chatbot interventions were efficacious across a range of populations and age groups, with both short- and longer-term interventions, and chatbot only and multicomponent interventions being efficacious.