Many affected counties have had experienced a shortage of personal protective equipment (PPE) during the coronavirus disease (COVID-19) pandemic. We aimed to investigate the needs of healthcare professionals and the technical difficulties faced by them during the initial outbreak. A cross-sectional web-based survey was conducted among the healthcare workforce in the most populous cities from three Latin American countries in April 2020. In total, 1,082 participants were included. Of these, 534 (49.4%), 263 (24.3%), and 114 (10.5%) were physicians, nurses, and other professionals, respectively. At least 70% of participants reported a lack of PPE. The most common shortages were shortages in gown coverall suits (643, 59.4%), N95 masks (600, 55.5%), and face shields (569, 52.6%). Professionals who performed procedures that generated aerosols reported shortages more frequently (p<0.05). Professionals working in the emergency department and primary care units reported more shortages than those working in intensive care units and hospital-based wards (p<0.001). Up to 556 (51.4%) participants reported the lack of sufficient knowledge about using PPE. Professionals working in public institutions felt less prepared, received less training, and had no protocols compared with their peers in working private institutions (p<0.001). Although the study sample corresponded to different hospital centers in different cities from the participating countries, sampling was non-random. Healthcare professionals in Latin America may face more difficulties than those from other countries, with 7 out of 10 professionals reporting that they did not have the necessary resources to care for patients with COVID-19. Technical and logistical difficulties should be addressed in the event of a future outbreak, as they have a negative impact on healthcare workers. Clinical trial registration: NCT04486404.