The upstream movement of juvenile eels (median total length (TL) = 138 mm) in the Mondego River, Portugal, was analysed between January 2017 and August 2019. A total of 12,019 individuals (TL ranging from 60 to 287 mm) were counted and measured on an eel ladder at Coimbra weir, 44 km upstream from the river mouth, during their upstream movements. Although eels were counted throughout the year, a clear seasonal peak was detected in spring and late summer, with 93% of total movements occurring between April and September. No differences were found in the total length of individuals moving upstream between years. Generalised additive models, with an explained deviance of 83% and an adjusted R2 of 0.88, identified minimum water temperature as the main environmental variable explaining these movements, with a threshold of 13 °C triggering the onset of movements, and a peak between 15 and 19 °C, which corresponds to the spring and summer periods. Other predictors such as photoperiod, precipitation and river flow also contributed as explanatory variables in the model but less importantly. The present results may contribute to the management of this endangered species in the southern region of its distribution range by defining adequate temporal windows to monitor and promote the movement of juvenile European eels through obstacles to migration.