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Aims: To gather data on agricultural practices in organic farms in Sao Paulo, Brazil, and evaluate their relationship with the microbiological characteristics of samples collected along the production chain. Methods and Results: Practices data were based on field observations and interviews with farmers in 10 selected organic lettuce producing farms. Counts of Enterobacteriaceae and surveys for Salmonella were performed in samples of lettuce (before and after washing), fertilizers, irrigation and washing water, all collected in the same farm. Water samples were also tested for total coliforms and generic Escherichia coli. Isolated Enterobacteriaceae were identified by MALDI‐TOF MS. Contamination of lettuce was influenced by some agricultural practices: chicken manure‐based fertilization resulted in higher Enterobacteriaceae counts in lettuce when compared to other types of manure, whereas pre‐washed lettuces presented lower microbial counts than non‐pre‐washed samples. Salmonella was detected in one lettuce sample by qPCR. Escherichia coli was detected in all irrigation water samples. All sample types contained Enterobacteriaceae species commonly reported as opportunistic human pathogens. Conclusions: The data highlight the need for improvement in the good agricultural practices in the studied farms. Significance and Impact of the Study: This study provides information on agricultural practices and microbiological characteristics of organic lettuce, contributing to the development of more accurate risk assessments. [ABSTRACT FROM AUTHOR]