Mammal species provide vital environmental and economic functions although they globally face numerous threats. Knowledge of the composition and diversity of mammal species provide information for appropriate management interventions. We conducted this study from July 2019 to February 2020 to assess the species composition, diversity, and conservation of medium- and large-sized mammals in the Asimba Forest Priority Area, northern Ethiopia. Data were collected through direct and indirect evidence from three habitat types, namely, natural forest, bushland, and plantation forest. We analyzed the data using descriptive statistics and the Species Diversity Index. We also used χ2 to compare differences in the number of observations and species richness of the mammal species in different habitats between the dry and wet seasons. A total of 263 observations were compiled, and 15 medium- and large-sized mammalian species were recorded in the Asimba Forest Priority Area. The Species Diversity Index and Evenness of the area were H′ = 2.436 and J = 0.899, respectively. Hamadryas baboon (Papio hamadryas) was the most commonly observed species, with 20.7 and 19.2 relative frequencies in the dry and wet seasons, respectively, whereas striped polecat (Ictonyx striatus) was the rarest species, with 3.0 and 0.0 relative frequencies during the dry and wet seasons, respectively. The number of observations (χ2 = 2.298, df = 2, and p=0.316) and species richness (χ2 = 0.115, df = 2, and p=0.943) did not differ significantly in the three habitats. Anthropogenic factors, mainly habitat destruction and overgrazing, were widely observed in the Asimba forest as conservation threats to the mammal species. The current study helps to increase knowledge about the mammal species of the area and is essential for their future conservation strategies.