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Healthcare workers worldwide present a high prevalence and incidence of low back pain due to manual patient handling tasks. Although education encourage the use of safe patient handling procedures, there is a lack of research about safe patient handling knowledge in developing countries. Therefore, the purpose of this study was to examine both levels of knowledge and factors associated with a good knowledge score among healthcare workers of a developing country.This was a cross-sectional study with 292 healthcare workers from the University Hospital of Botucatu Medical School. A knowledge score was generated based on participant responses to a structured questionnaire, with a score of 70% or higher indicating good knowledge. Frequency statistics and logistic regression were conducted to analyse the data.The majority of participants did not achieve a good level of knowledge (81,5%). Only 54,1% were aware of the high risk presented by patient-handling tasks. Most participants (84,8%) were not knowledgeable of the recommended maximum weight limit for use in patient-handling tasks, 62,3% and 53,4% answered that relying on proper body mechanics and that the use of back belts are effective prevention methods, respectively. The knowledge gap was most prominent in the domain of safe patient handling, only 2,4% of participants answered correctly all questions regarding this topic. Regarding the associated factors towards a good level of knowledge, the only significant relationship in univariate as well as multiple logistic analysis was level of education, being less educated – elementary or high school education – was a significant predictor of lower levels of knowledge (OR=4.776, 95% CI: 1.056 to 21.600).A substantial knowledge deficit was identified among healthcare workers in our study. Policies and education campaigns to increase knowledge and improve preventive practices should be developed.