BACKGROUND In Chile, members of the civil society and government achieved the passing of the HIV/AIDS Law (19.779). The level of knowledge of the law held by healthcare workers in Chile is not well known. AIM To analyze the effect of an intervention on knowledge of the existence of the law and its application in clinical practice among primary healthcare workers in southeastern Santiago. MATERIAL AND METHODS Healthcare workers of primary care centers were invited to participate in the study. One group received an educational intervention lasting a total of 16 hours, about AIDS physiopathology, sexually transmitted diseases, communication with patients and current legislation. A control group did not receive the educational intervention. Both groups answered a self-administered questionnaire about the HIV/AIDS law at baseline and three months after the intervention. RESULTS The intervention was carried out in 262 workers and 293 participated as controls. The initial evaluation revealed that only 16.3% (n = 89) had heard of the law, without any significant difference between intervention and control groups. The knowledge about the law improved by 65% in the intervention group and did not change in controls. At the end of the education period, the intervention and control groups improved their global knowledge by 29 and 3%, respectively (p < 0.05). CONCLUSIONS The educational intervention was effective in improving knowledge of the HIV/AIDS Law among Chilean healthcare workers.