Background and Objectives: Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by challenges in communication, social interactions, and repetitive behaviors. Although the factors that influence the development of this condition are unknown, certain chemical compounds such as pesticides have been proposed as possible contributors. Due to the lack of an established causal link between pesticide exposure and ASD, this study aimed to evaluate this potential association. Materials and Methods: A case-control study was carried out to ascertain the prevalence and risk associated with ASD in relation to pesticide exposure over a 21-year study period (2000–2021). Results: We included 2821 individuals diagnosed with ASD residing in areas of both high and low pesticide exposure in southern Spain. There was a rise in the ASD prevalence rate in regions with elevated pesticide use when compared to regions with low use [odds ratio (OR): 1.34, 95% confidence interval (CI), (1.24–1.44)]. Notably, men had the highest likelihood, with an OR: 1.42, 95% CI, (1.30–1.55). Furthermore, after performing multiple binary logistic regression adjusted for age, sex, and geographical area, males exhibited a higher likelihood compared to females [OR: 2.41, 95% CI, (2.21–2.62)]. Conclusions: Overall, this research suggests a connection between heightened environmental pesticide exposure due to increased agricultural use and autism.