In heterocyclic chemistry, many studies have adopted the synthesis of novel benzotriazinone derivatives, as they have shown significant potential in biomedical research, particularly as enzyme inhibitors. This makes benzotriazinone a valuable scaffold for the development of new pharmaceuticals. Herein, a novel series of benzotriazinone derivatives was successfully synthesized, followed by docking studies. Ethyl 2-carboxyphenylazocyanoacetate was synthesized through the reaction of one mole of diazonium salt with a percentage of a mole of ethyl cyanoacetate in an alkaline medium. One of the derivatives, Triazine 12, exhibited promising in silico antitumor activity, according to molecular docking studies, which was then confirmed in vitro by determining its cytotoxicity against the human tumor cell lines HepG-2 and MCF-7, with IC50 values of 78.53 ± 3.49 µg ml−1 and 48.31 ± 2.37 µg ml−1, respectively, in addition to low cytotoxicity against normal lung fibroblast cells (MRC-5), with a moderate antioxidant capacity shown in the DPPH radical scavenging assay. Furthermore, its suitability for radiolabeling as a tracer for in vivo studies was checked. Triazine 12 was directly radiolabeled with technetium-99m (99mTc), yielding a radiochemical purity of 95.4% ± 0.46%. A biodistribution study in tumor-bearing mice of [99mTc]-labeled Triazine 12 exhibited significant tumor targeting properties with positive T/NT ratios, reaching a peak of 4.65 (more than many tumoral radiopharmaceuticals) 1-hour post-injection, which highlights its potential as a novel radiotracer for tumor imaging.