The annual cultivation pattern in the Uma-oya catchment in Sri Lanka is characterized by Yala and Maha rainfall periods and associated cropping. Two cultivation seasons were compared for pesticide residues: base flow, field drainage, and the runoff and supplementary sediment data for three sites in the catchment. Organophosphate and N-methyl carbamate pesticide analysis confirmed a higher concentration in the Yala season with low-flow conditions. Acetylcholinesterase (AChE) activity was measured by standard spectrometry in the brain, muscle, and eye tissues of three freshwater cyprinid fishes, Garra ceylonensis, Devario malabaricus, and Rasbora daniconius from three study sites during months overlapping two seasons in 2010 (December) and 2011 (July). Baseline AChE data were measured from fish samples from a forested reserve in the Knuckles. A 73% inhibition in muscle AChE activity in G. ceylonensis was associated with intense pesticide exposure months in the Yala season. The AChE inhibition more than 70% in G. ceylonensis eyes in both Yala (76%) and Maha (72.5%) seasons indicates particular sensitivity of eye tissue to inhibitors. The less dramatic AChE inhibition in the eye tissues in D. malabaricus and R. daniconius in both seasons indicates exemplary protective capacity of muscle AChE in fish. The highest inhibition of AChE (up to 60% in brain and up to 56% in muscle AChE activity in R. daniconius and up to 47.8% in brain and up to 64.6% in muscle AChE activity in D. malabaricus) occurred during the Yala season. Tissue AChE activity and physiological activity in fish were correlated. The results collectively indicate that AChE is a consistent biomarker for diffused contaminant exposure in agricultural catchments.