Freshwater ecosystems are highly threatened due to increased population growth, thereby affecting biodiversity. The objective of designating an area as ‘protected’ is to reduce habitat degradation and biodiversity loss. However, most protected areas focus on the conservation of the terrestrial environment and its related biodiversity. Little attention is given to the protection of freshwater biodiversity and consequently far less is known on how freshwater biodiversity differs between wetlands with a different protection status. The objectives of this study was to investigate alpha and beta diversity of macroinvertebrates and wetland dependent birds in wetlands with a different protection status and thus a different level of human impact in southwestern Ethiopia. Data on macroinvertebrates, birds, physico-chemical water quality, human disturbance and vegetation cover were collected from 42 sampling sites during dry and wet season of 2015. Besides the calculation of alpha and beta diversity, multiple regression models were used to identify the main drivers of the variation in beta diversity of macroinvertebrates and birds in these wetlands. Our results revealed that the average alpha diversity of both macroinvertebrates and wetland dependent birds was significantly higher in protected wetlands (low overall human disturbance) compared to unprotected wetlands (high overall human disturbance), whereas beta diversity was higher in unprotected wetlands for wetland dependent birds. Turnover contributed more than nestedeness to total beta diversity for both macroinvertebrates and wetland birds in protected and unprotected areas. Human disturbance, water temperature, pH and dissolved oxygen were significantly related to beta diversity of macroinvertebrates, whereas beta diversity of wetland dependent birds was significantly related to vegetation cover, nutrients and chemical oxygen demand. Conservationists should give attention to both alpha and beta diversity of freshwater wetlands in order to conserve wetland biodiversity maximally.