Objectives Unregulated use of pesticides continues in developing countries in the presence of illiteracy and limited safety training and practices. This paper describes training and safety practices when mixing and spraying pesticides, and acetylcholinesterase levels among women farmers in KwaZulu-Natal, South Africa. Methods A cross-sectional study conducted in women working in small-scale agriculture in rural KwaZulu-Natal, South Africa in 2006 assessed pesticide training and safety practices using a questionnaire survey and measured acetylcholinesterase levels in 803 women. Three components of safety behaviour were identified through principle component analysis and described. Results The mean age of participants was 41.8 years (range 18-82 years) with a mean of 6.9 years (range 1-12 years) of education among school attendees. Among the 803 women included, 366 (45.6%) were the primary sprayers on their farm. Only 16.4% of the sprayers had ever attended a pesticide training course and only 18.0% reported ever reading pesticide labels. Of the women using some form of protective equipment, 56.7% and 54.9% reported doing so when mixing and spraying pesticides, respectively. The mean acetylcholinesterase level corrected for haemoglobin among study participants was 28.9 U/g Hb (95% CI 28.4 to 29.4). Conclusion Women working in small-scale agriculture in rural KwaZulu-Natal with limited access to pesticide training observe few safety practices when mixing and spraying pesticides.