Objective: To evaluate COVID-19 knowledge, attitudes, and practices among health care workers (HCWs) practicing in Latin American countries during the first surge of the COVID-19 pandemic. Methods: This was a multinational cross-sectional survey study, using an online self-administered questionnaire. The final version of the questionnaire comprised 40 questions, organized in five sections: demographic and professional characteristics; COVID-19 knowledge; attitudes toward COVID-19; COVID-19 practices; and institutional resources. Results: The study involved 251 HCWs from 19 Latin American countries who agreed to participate. In our sample, 77% of HCWs participated in some sort of institutional training on COVID-19, and 43% had a low COVID-19 knowledge score. COVID-19 knowledge was associated with the type of health center (public/private), availability of institutional training, and sources of information about COVID-19. Concerns about not providing adequate care were reported by 60% of the participants. The most commonly used ventilatory strategies were protective mechanical ventilation, alveolar recruitment maneuvers, and prone positioning, and the use of drugs to treat COVID-19 was mainly based on institutional protocols. Conclusions: In this multinational study in Latin America, almost half of HCWs had a low COVID-19 knowledge score, and the level of knowledge was associated with the type of institution, participation in institutional training, and information sources. HCWs considered that COVID-19 was very relevant, and more than half were concerned about not providing adequate care to patients.