This book constitutes the proceedings of the Second Australasian Conference on Artificial Life and Computational Intelligence, ACALCI 2016, held in Canberra, ACT, Australia, in February 2016. The 30 full papers presented in this volume were carefully reviewed and selected from 41 submissions. They are organized in topical sections named: mathematical modeling and theory, learning and optimization, planning and scheduling, feature selection, and applications and games.