The excessive application of various synthetic pesticides led to control difficulties, including insect resistance and environmental contamination. This study aimed to evaluate the insecticidal and repellent activities of twelve botanical powders and aqueous extracts against Tribolium castaneum, with a focus on acetylcholinesterase and glutathione-S-transferase detoxification enzyme activities. Toxicity tests revealed that Azadirachta indica dry powder was the most harmful, having the lowest LD50 value of 2.09% w/w, while in aqueous extract A. indica was the most toxic, with an LC50 of 2.20% after 24 h. Repellency tests demonstrated that A. indica exhibited the highest repellent effect in both powder and aqueous forms (86.66%). As a result of the most effective botanical application, biochemical analyses showed that acetylcholinesterase activity remained highest (6.17 ± 0.17 U/min/g) in A. indica-treated insects, whereas glutathione-S-transferase enzyme activity peaked in response to Eucalyptus tereticornis (85.00 U/min/g), indicating a strong physiological defense response. These results indicate that plants like A. indica, D. stramonium, E. tereticornis, and Ar. nilagirica can be used as promising bio-insecticides options for controlling T. castaneum in stored products.