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The life history pattern and feeding ecology of introduced Gambusia holbrooki was studied in Lake Pamvotis, a shallow lentic ecosystem. The length frequency analysis identified two different age groups. The first recruits were recorded in July and were present thereafter until December. Females reached greater sizes, grew faster and lived longer than males. The largest sizes found were 43.06 mm for females and 34.34 mm for males. Sex-ratio deviated significantly from unity (1.65:1). Multiple spawning occurred throughout the reproductive cycle (from May to August). The average brood size per female reached its maximum in June (50.8 embryos per female). After reproduction, the percentage of parental individuals decreased drastically. Mosquitofish fed mainly on zooplankton (Copepoda, littoral Cladocerans), but surface insects (such as Nematoceran adults, basically Chironomid, collembolans, Hemiptera, spiders and worms) were additional food. Diet composition showed little seasonal variation. Trophic niche breadth was largest in summer (H=0.68), and smallest in autumn (H=0.46). The mean volume of stomach contents increased significantly for fish larger than 30 mm TL (Total Length). Gambusia holbrooki showed high intrapopulation dietary overlap and followed a specialist feeding strategy with adaptation to seasonal prey availability. Moreover, Culicidae larvae constituted a small quantitative fraction of the G. holbrooki diet which raised questions about the extensive use of mosquitofish for biological control of mosquitoes.