The first analyses of biological components in glaciers of the Chilean lake district are presented based on microalgae biovolume, pollen and other microorganisms detected in shallow (∼10 m) firn/ice cores. Three cores were retrieved, two at Volcán Mocho-Choshuenco (39°55′ S, 72°02′ W; summit at 2422 m a.s.l; east glacier at 2000 m a.s.l.), and one at the summit of Volcán Osorno (41°06′ S, 72°30′ W; 2652 m a.s.l.). Microalgae, protozoa and pollen quantified in the samples obtained from the two summit cores show clear fluctuations interpreted as seasonal signs. In contrast, δD and many chemical species from the summit cores show strong dampening at depth, probably due to water percolation. The limited information provided by isotopic and chemical analyses is used to support the seasonal interpretation of biological parameters from the summit cores, with microorganism maxima inferred to occur in summer and pollen maxima in spring. A good comparison is found between mass-balance estimations from the Volcán Mocho-Choshuenco summit core and values obtained near that site by means of the stake method. It is concluded that biological analyses of firn/ice cores provide reliable estimations of annual and seasonal markers from these temperate glaciers.