Diarrhoeal diseases are a common cause of morbidity and are associated with mortality in HIV-infected populations. Little is known about the contribution of clinical and socio-environmental factors to the risk of diarrhoea in these populations in rural sub-Saharan Africa. We conducted a case-control study of people attending a rural HIV clinic with an episode of diarrhoea in Bushbuckridge, South Africa. Cases were defined as HIV-positive adults with symptoms of diarrhoea before or after initiation of antiretroviral therapy (ART). Controls without diarrhoea were randomly selected from clinic attendees. Structured questionnaires and case-file reviews were undertaken to describe clinical and socioenvironmental risk factors. We recruited 103 cases of diarrhoea from 121 patients meeting case definitions. Cases were more likely to be women (P = 0.013), aged over 45 years (P = 0.002), divorced or separated (P = 0.006), have limited formal education (P = 0.003), have inadequate access to sanitation facilities (P = 0.045), have water access limited to less than three days per week (P = 0.032) and not yet initiated on ART (P < 0.001). In multivariate analysis, diarrhoea remained associated with female gender (adjusted odds ratio [aOR]: 2.02, 95% CI 1.10-3.73), older age (aOR: 6.31, 95% CI 1.50-26.50), limited access to water (aOR: 2.66, 95% CI 1.32-5.35) and pre-ART status (aOR: 5.87, 95% CI 3.05-11.27). Clinical and socio-environmental factors are associated with occurrence of diarrhoeal disease among rural HIV patients in South Africa. Further intervention research is urgently needed, combining community- and clinic-based approaches, to improve access to water, sanitation and ART for rural areas with high HIV prevalence, along with structural interventions to address gender inequities.