Background: Organophosphate (OP) pesticide poisoning remains a critical public health issue in developing countries, including India, Pakistan, and Sri Lanka. These compounds, used extensively as insecticides, petroleum additives, and nerve agents, continue to pose significant health risks due to their widespread availability and limited public awareness. This study aims to assess the utility of biochemical biomarkers in evaluating the severity of OP poisoning. Materials and methods: An observational study at PES Institute of Medical Sciences and Research (PESIMSR) from January 2021 to June 2022 included 152 patients with confirmed OP poisoning. Exclusion criteria included co-existing conditions and recent interventions. Biochemical markers—serum amylase, plasma cholinesterase (CHE), and serum creatine kinase (CPK)—were analyzed at admission, and on Days 1, 3, and 5. Clinical outcomes were recorded, including ventilator support and mortality. Data were analyzed using IBM SPSS Statistics. Results: Patients were predominantly aged 31-40 years, with a male predominance (1.86:1). Of the 152 patients, 127 survived, and 25 died. Elevated serum amylase, CPK, and decreased CHE levels were associated with worse outcomes. Significant correlations were observed between high amylase and CPK levels with poor prognosis and increased need for ventilator support. The mean APACHE II score was higher in patients needing ventilation. Conclusion: Biochemical biomarkers, particularly serum amylase, CPK, and plasma CHE, effectively assess OP poisoning severity. Elevated levels are linked to higher mortality and ventilator dependency, underscoring their utility in managing OP poisoning.