Anthropogenic habitat alteration is considered to be one of the major culprits of biodiversity loss worldwide. This paper presents an investigation on the response of tropical amphibian assemblage to a gradient of habitat alteration in Rajah Sikatuna Protected Landscape (RSPL) of Bohol, Philippines. The abundance, richness, diversity and composition of amphibians were compared among the different habitat types (lower montane forest, dipterocarp forest, riparian forest, grassland and agricultural areas), as well as their relationships with environmental or habitat characteristics were examined. A total of 318 amphibian individuals from 18 species, and belonging to 7 families were recorded. It was found that amphibian abundance, richness and diversity differed significantly between habitat types where it was generally higher in less disturbed habitats (e.g., forests and riparian) compared to highly altered habitats (e.g. grassland and agricultural areas). In relation to species composition pattern, forests and riparian habitats were strongly associated with rainforest and stream-dependent amphibian species while highly altered habitats such as grassland and agricultural areas were associated with those that were open-habitat specialist and disturbance tolerant species. Moreover, the most important environmental variables influencing the pattern of amphibian diversity in the area included grass cover, litter depth, ground relative humidity, temperature, and elevation. In summary, the assemblage of amphibians in the RSPL, Bohol was affected by habitat alteration, highlighting the importance of the sustained protection efforts of the landscape.