Background The study examined how the diagnostic radiopharmaceutical 99mTechnetium-sestamibi (99mTc-MIBI) affects in vivo biochemical parameters in Rattus norvegicus (Berkenhout, 1769). Given the widespread use of 99mTc-MIBI in nuclear medicine for imaging cardiac and oncological conditions, understanding its potential metabolic and biochemical impacts is crucial. Objective The study examines the influence of 42.7 MBq (1.15 mCi) of 99mTc-MIBI on biochemical parameters in Rattus norvegicus (Berkenhout, 1769). Methods The research utilized Wistar strain Rattus norvegicus specimens that were about 3 months old with an average weight of 180 g. The Department of Biology at the Faculty of Science, University of Sarajevo vivarium bred the rats in purpose-built cages that fully adhere to experimental animal standards. The animals were divided into a control group and two experimental groups, which received 42.7 MBq of 99mTc-MIBI, followed by heart punctures performed at 2 h and 72 h after administration. The control group (K) composed of five males while experimental group 1 (G1) contained one male along with two pregnant females and two non-pregnant females. Five female animals were part of the second experimental group 2 (G2). Results Overall, the analysis indicated significant differences in urea levels between the groups, while glucose levels did not show significant differences. Creatinine levels exhibited some significant variance as well, particularly between groups K and G2. Conclusions Our in vivo studies demonstrated that biochemical parameters changed following the administration of 99mTc-MIBI. The tested organism displayed metabolic changes that suggest either a stress response or toxicological effects.