This study aimed to investigate the natural feeding behavior of Nile tilapia in Lake Langeno, Ethiopia, with emphasis on potential spatial, size and seasonal effects on ingested food items. This study of the food and feeding biology of O. niloticus in Lake Langeno, Ethiopia, was conducted from March 2016to February 2017. Fish samples were collected monthly from six different sampling sites using different mesh sizes of gillnets. A total of 610 fish specimens with full stomachs were considered for the assessment of feeding biology. In total, seven food items, namely phytoplankton, zooplankton, insects, detritus, macrophytes, fish parts and nematodes, were identified from the fish stomach contents. Phytoplankton was the most commonly consumed food prey, followed by detritus, zooplankton and macrophytes. The other food items were occasionally and randomly consumed. Phytoplankton and detritus were the dominant food prey in the dry season, with zooplankton and macrophytes the main prey during the wet months. The contribution of phytoplankton, zooplankton and insects were slightly highest in small-sized groups (<10 cm), whereas detritus, macrophytes and fish parts were highest in larger-size groups (>20 cm) (p < 0.05). The present results point to a concurrence of the relative importance of dietary items at the individual level, species level and among the study sites. Phytoplankton was the primary consumed food item, which indicates the specialist feeding strategy of Nile tilapia in the lake. Generally, food items of plant origin, typically associated with less protein content than animal origin food items, dominated the stomach contents of Nile tilapia. The dietary pattern of Nile tilapia in Lake Langeno shifts with size and season, aspects that might warrant further study in view of aquaculture applications as well as climate change.