Fatal motor vehicle crashes (MVCs) continue to be a common occurrence worldwide. This paper presents a retrospective analysis of the toxicological investigation of drivers and motorcyclists fatally injured in MVCs in Scotland from 2012 to 2015. One hundred and eighteen cases with full toxicological analysis, i.e., alcohol, drugs of abuse and prescription drugs, were examined. Of those 118 MVC cases, 74 (63%) were car drivers, 32 (27%) were motorcyclists and the remaining were drivers of other vehicles such as large goods vehicles. The majority of deceased drivers and motorcyclists were male (N = 104, 88%). For the toxicological findings, 51 (43%) of the cases were negative, and of the 67 (57%) positive cases, alcohol and cannabinoids were the most frequently detected substances, followed by opioids and benzodiazepines. Fifteen percent of all drivers and motorcyclists were over the prescribed blood alcohol limit at the time of analysis. In comparison to previous reports of drug use by drivers in Scotland, benzodiazepines and new psychoactive substances were less common findings in fatally injured drivers and motorcyclists than in drivers suspected of being impaired.