Kala azar occurs among seasonal and migrant agricultural workers in northwest Ethiopia and accounts for almost 60% of the disease burden in the country. We conducted a quantitative study on the level of knowledge and practice of this vulnerable group in relation to kala azar transmission and acceptability of its vector control tools. A total of 403 workers were randomly selected from eight farms using a purposive sampling technique. Knowledge and practice scores were calculated based on 12 and 9 core questions, respectively. Binary logistic regression was used to identify factors associated with knowledge and practice. A large gap in knowledge of the disease and the vector was evident with 61.8%, 24.6%, and 13.6% of the workers having poor, moderate, and good levels of knowledge scores, respectively. Similarly, 95% of the seasonal workers reported poor level of use of protective measures against the bite of the sand fly vector. Good level of knowledge about kala azar and its sand fly vector was statistically associated with formal education (adjusted odds ratio [AOR] = 2.11; 95% CI = 1.17, 3.80; < 0.05) and previous exposure to health education (AOR = 4.72; 95% CI = 1.99, 11.16; < 0.001). Despite poor current knowledge and practice, a large proportion of the study participants showed interest in using vector control tools if made available, with 78% of the seasonal and migrant workers expressing some willingness to pay for different measures that can protect against sand fly bites. Therefore, we strongly recommend that comprehensive health education and vector control programs should be provided to these workers.