Introduction: Nuclear medicine services, though still an emerging area in the field of medicine in Nigeria, have not been optimally utilised, possibly due to a lack of awareness by health workers. This study evaluated the knowledge and awareness of pharmacists and patients regarding radiopharmaceutical use in disease management. Methods: In this cross-sectional survey in southwest Nigeria, online self-administered and interviewer-administered structured questionnaires were used to collect data on the knowledge and perception of radiopharmaceuticals from 343 pharmacists and 67 ambulatory patients. The influence of the participant’s socio-demographic characteristics on the knowledge of radiopharmaceuticals scores was assessed with Man-Whitney U and Kruskal Wallis tests at p < 0.05. Results: The study found that 191 (55.7%) pharmacists and 75 (98.7%) patients had good knowledge of radiopharmaceuticals, while 70 (21.3%) pharmacists knew radiopharmaceuticals are regulated as radioactive substances and drugs. Most patients, 47 (61.8%), knew how to prevent radiation exposure to others, and 68 (89.5%) believed radiopharmaceuticals would help disease management. Conclusion: Pharmacists and patients have good knowledge of the use of radiopharmaceuticals. Pharmacists’ knowledge was influenced by the state of practice and prior knowledge of radiopharmaceuticals, while the number of clinics attended influenced patients’ knowledge.