<p>Aphaniidae killifish represent a group of small-bodied teleosts that inhabit a wide range of transitional and extreme aquatic environments. Their remarkable adaptability, ecological niche, and high levels of endemism make them valuable models for the investigation of biodiversity patterns, evolutionary processes, and conservation strategies in fragmented and vulnerable habitats. Drawing from case studies across the Mediterranean Sea and the Middle East, this research examines how ecological plasticity, reproductive strategies, and molecular tools allow to interpret the resilience patterns of the distribution. Our phylogenetic analyses confirm their complex evolutionary relationships and highlight the role of geological and biogeographical events in shaping their diversification. The potential of Aphaniidae as comprehensive models that connect laboratory insights and ecosystem-level dynamics is highlighted by the integration of genetics, physiology, and field ecology. Encouraging their use in biodiversity research and conservation efforts offers a crucial chance to enhance our capacity to monitor, understand, and mitigate biodiversity loss in rapidly changing aquatic systems. This review synthesizes advances in understanding the evolutionary adaptations, ecological roles, reproductive strategies, and (eco) toxicological applications of killifish. It highlights their potential to bridge the gap between laboratory models and real-world ecological processes, advocating for their broader adoption in scientific research. In this regard, killifish represents an essential resource for addressing global issues of biodiversity conservation, ecosystem management, and environmental resilience.</p>