The two-volume set LNCS 16223 + 16224 constitutes the refereed proceedings of the 16th International Conference on Decision and Game Theory for Security, GameSec 2025, which took place in Athens, Greece, in October 2025. The 35 full papers and 3 short papers included in the proceedings were carefully reviewed and selected from 51 submissions. They were organized in topical sections as follows: Part I: Game-theoretic foundations and learning; game-theoretic cybersecurity frameworks; deception and adversarial defense; AI and LLMs in security; Part II: Strategic defense and robustness; applicatiouns in security and networks; emerging threats and anomaly detection.