Brucellosis is a neglected infectious disease caused by animals and is becoming a public health problem in developing countries. There are limited case reports of human brucellosis in Indonesia because the symptoms are not specific. This study investigated the association between serological tests for brucellosis among workers and their knowledge, attitude, and behavior. The study was conducted on farmworkers in a dairy cattle milking center in Boyolali and Cilawu. A total of 149 respondents, according to the inclusion criteria, were included. Around 3 ml of blood was collected from the respondents, followed by structured interviews to determine the level of knowledge, attitudes, and behavior towards brucellosis in humans. The sera were tested with the rose bengal test (RBT) and complement fixation test (CFT). Results showed that 3.3% of respondents were positive for RBT in Boyolali and 7.01% in Cilawu. However, CFT showed that 5.3% of respondents were positive in Cilawu, but 0% in Boyolali. The level of knowledge and attitudes of respondents regarding brucellosis in humans was still lacking, and the behavior of respondents on the farm was mostly good, in the sense of understanding the procedures for maintaining healthy dairy cattle correctly, but maintaining personal health was still lacking. This study underscores the need for immediate action to improve the situation. Public health professionals, researchers, and policymakers have a crucial role in implementing regular health education programs for farmworkers and improving the availability of personal protective equipment.