The study aimed to assess the association of long-term pesticide exposure (5 years) with hematological, serum biochemical parameters and acetylcholinesterase activity in farm workers. These pesticides included organophosphorus pesticides, carbamates, pyrethroids, dithiocarbamates, and other pesticides such as endosulfan. Applying a cross-sectional study design, 69 females from a pesticide-exposed farm population and 30 females from a district not using pesticides (reference group) were studied. The mean red cell corpuscular volume and hematocrit values were significantly lower (74.7 9.1 fl; 95% CI 72.5–76.9 and 32.0% 4.6%; 95% CI 30.9–33.1, respectively) in the exposed compared to the reference group, whereas mean corpuscular hemoglobin concentration and platelets were significantly higher (37.4 3.8 g/dL; 95% CI 36.5–38.3 and 374.1 95.3/L; 95% CI 351.2–396.9, respectively) in the exposed compared to the reference group. Mean serum glutamic oxaloacetate transaminase (20.7 8.9 U/L; 95% CI 18.5–22.9) and creatinine (83.9 6.6 mol/L; 95% CI 82.3–85.5) were significantly higher in the exposed compared to the reference group. A higher mean esterase activity (AChE 0.6 0.2 mM/min/mg protein; 95% CI 0.56–0.7; BChE 0.9 0.4 mM/min/mg protein; 95% CI 0.9–1.1) was noted in the exposed group. Regression models suggest that occupational exposure (p < 0.001) could be a predictor of esterase (AChE and BChE) activity and biochemical changes (= 0.4, 95% CI: 0.3–0.5; = 0.7, 95% CI: 0.6–0.9, respectively). Long-term pesticide exposure affects the hemato-biochemical and esterase responses, establishing the need for further studies.Research Council of Norway (NFR nr: 204051)