Appraisal of tree and fauna species is crucial; this ensures effective conservation of the forests and their resources. This study aims at an appraisal of tree and fauna species diversity in Osun-Osogbo Sacred Grove, Nigeria for record update, knowledge, and monitoring species status for conservation management. A total of 70 tree species belonging to 26 families were recorded. The tree species in the Fabaceae and Apocynaceae families were the most abundant. Tree species richness indices for the three zones were 2.16, 1.89, and 1.72, respectively. Most of the tree species encountered were in the IUCN categories of vulnerable (6.7%), least concerning (71.1%), and not evaluated (71.1%). The result revealed that the core zone was richer and more diverse than the buffer and the outer zones, with diversity indices of 3.06, 2.995, and 2.16, respectively. The fauna species diversity and richness index were 3.40 and 2.35, respectively. The least vulnerable fauna species were 86.4%, with 6.8% vulnerable. Class Aves has the highest percentage of 61.4%, with Gastropoda being the least-represented (2.3%). Mona monkeys were the most sighted fauna species in the area. Thus, there is an urgent need to increase the level of security around the sacred grove and to ensure sustainable management strategies are fully implemented in the study area.