We studied the variations in life history and feeding ecology of G. holbrooki in Acigol, a groundwater-dependent wetland and the only distribution area of the critically endangered Aphanius transgrediens. Fish were sampled from three sites with different ecological characteristics: Site 1 with stable conditions, small size and high fish density; Site 2 with stable conditions, large size and medium fish density; Site 3 with variable conditions, large size and low fish density. The scale readings showed two age groups in males and three in females. The reproduction period was from April to September. More embryos and heavier gonads were found in specimens at Site 3. The overall ratio of fertilised eggs and the absolute and relative fecundities were the lowest at Site 1. Two different trophic positions, plant-based omnivory and animal-based omnivory, were detected. The main food items at Site 2 and Site 3 were adults and larvae of Diptera, as well as zooplankton. At the same time, mosquitofish fed predominantly on plant sources and occasionally on the other ones at Site 1. Significant temporal and ontogenetic differences in feeding were also determined. The highest diet diversity and broadest trophic niche were found at Site 2.