Injury deaths have a major impact on public health systems, particularly in the Latin American region; however, little is known about how different drugs, in combination or not with alcohol, interact with each injury type. We tested an epidemiological protocol for investigating alcohol and other drug acute use among fatally injured victims taking into account the injury context for all injury causes in Sao Paulo, Brazil. Blood alcohol and drug content were fully screened and confirmed following a probability sample selection of decedents (n = 365) during 19 consecutive months (2014-2015). Drug concentrations, including benzodiazepines, cannabis, cocaine, and opioids were determined by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography tandem mass spectrometry (LC-MS/MS). Toxicology data were interpreted in combination with injury context retrieved from police records regarding cause, place of injury, and victims' criminal history. More than half of all fatally injured victims studied were under the influence of at least one substance (55.3%). Alcohol was the leading substance consumed before a fatal injury event (30.1%), followed by cocaine (21.9%) and cannabis (14%). Illicit drug use (cocaine and cannabis) comprised more than two thirds of all drug-related deaths. Alcohol-positive deaths are over-represented among road traffic injuries, while drug-positive deaths are more prevalent among intentional injuries. Victims who had previous criminal convictions were significantly more likely to have used illicit drugs compared to those who did not have a criminal background. We estimated that one in every two fatal injuries in the city of Sao Paulo is associated with acute substance use by the victim. The health burden attributed to alcohol- and drug-related fatal injury events has reached significant higher levels in Latin American cities such as Sao Paulo compared globally.