Importance: Prenatal exposure to certain medications has been hypothesized to influence the risk of autism spectrum disorders (ASD). However, the underlying effects on the neurotransmitter systems have not been comprehensively assessed. Objective: To investigate the association of early-life interference with different neurotransmitter systems by prenatal medication exposure on the risk of ASD in offspring. Design, Setting, and Participants: This case-control study included children born from January 1, 1997, through December 31, 2007, and followed up for ASD until January 26, 2015, within a single Israeli health maintenance organization. Using publicly available data, 55 groups of medications affecting neurotransmitter systems and prescribed to pregnant women in this sample were identified. Children prenatally exposed to medications were compared with nonexposed children. Data were analyzed from March 1, 2017, through June 20, 2018. Main Outcome and Measures: Hazard ratios (HRs) and 95% CIs of ASD risk associated with exposure to medication groups using Cox proportional hazards regression, adjusted for the relevant confounders (eg, birth year, maternal age, maternal history of psychiatric and neurologic disorders, or maternal number of all medical diagnoses 1 year before pregnancy). Results: The analytic sample consisted of 96249 individuals (1405 cases; 94844 controls; mean [SD] age at the end of follow-up, 11.6 [3.1] years; 48.8% female), including 1405 with ASD and 94844 controls. Of 34 groups of medications, 5 showed nominally statistically significant association with ASD in fully adjusted models. Evidence of confounding effects of the number of maternal diagnoses on the association between offspring exposure to medication and ASD was found. Adjusting for this factor, lower estimates of ASD risk among children exposed to cannabinoid receptor agonists (HR, 0.72; 95% CI, 0.55-0.95; P =.02), muscarinic receptor 2 agonists (HR, 0.49; 95% CI, 0.24-0.98; P =.04), opioid receptor κ and ϵ agonists (HR, 0.67; 95% CI, 0.45-0.99; P =.045), or α 2C -Adrenergic receptor agonists (HR, 0.43; 95% CI, 0.19-0.96; P =.04) were observed. Exposure to antagonists of neuronal nicotinic acetylcholine receptor α was associated with higher estimates of ASD risk (HR, 12.94; 95% CI, 1.35-124.25; P =.03). Conclusions and Relevance: Most of the medications affecting neurotransmitter systems in this sample had no association with the estimates of ASD risk. Replication and/or validation using experimental techniques are required.