Agricultural workers’ perceptions of irrigation water quality influence their water management practices, impacting food safety and public health. However, there are no known data on the perceptions of agricultural workers in Mbeya, Tanzania, regarding the chemical and microbial quality of irrigation water, as well as the associated health risks. To address this data gap, we conducted a cross-sectional survey from September to October 2024 among agricultural workers (n=171) in Mbeya, Tanzania. Descriptive statistics and logistic regression were performed using SAS and R. Awareness of the importance of irrigation water quality was high among agricultural workers, as 78% of agricultural workers agreed that poor water quality can negatively impact agricultural output, and 62% acknowledged poor irrigation water quality as a contributing factor to health problems in their communities. Men were more likely to report being knowledgeable about chemical contaminants (adjusted odds ratio [aOR] = 3.42, 95% CI: 1.68–6.96, p = <0.001) and microbial contaminants (aOR = 2.87, 95% CI: 1.41–5.83, p = 0.004) compared to women. The importance of regular water quality testing also differed by gender. Men were more likely than women to report that they understood the importance of water testing (aOR = 2.52, 95% CI: 1.26–5.04, p = 0.009). Our findings are the first of their kind from Mbeya, Tanzania, revealing significant gaps in agricultural workers’ understanding of chemical and microbiological contaminants in irrigation water and highlighting the need for targeted education and capacity-building initiatives to improve irrigation water quality management practices in this area.