Zooplankton biodiversity is deemed as a realiable indicator of water quality. For 40 years, the Guatemalan lake Amatitlán has shown signs of eutrophication, with measurable impacts on the local zooplankton diversity. Biotic and abiotic variables were surveyed at four sites of lake Amatitlán (Este Centro, Oeste Centro, Bahía Playa de Oro and Michatoya) in 2016 and 2017. The species richness and abundance of rotifers, cladocerans, and copepods were analyzed. The dynamic composition of zooplankton was studied and the system environmental parameters were analyzed in two seasons (rainy and dry season) for both years. Characteristical values of eutrophied tropical systems were obtained, with high rotifer diversity (11 species) and abundance. At present, the rotifers Brachionus havanaensis (109 ind/L) and Keratella americana (304 ind/L) were the most abundant species in the system. The copepod Mastigodiaptomus amatitlanensis considered as endemic in 1941 is absent nowadays, but we reported the unprecedented occurrence of two exotic copepods (i. e., Thermocyclops crassus, Mesocyclops thermocyclopoides) for lake Amatitán and all Guatemala. The presence of large zooplankton like adults and immature copepods (Arctodiaptomus dorsalis) and cladocerans (Ceriodaphnia sp.), in site “Este Centro”, indicates a relatively healthy community and represents a focal point for the conservation for this lake.