In recent years, traditional olive groves have undergone a shift towards modern farming practices, raising concerns about their potential impact on local fauna. Despite the documented effects of various agricultural practices on wildlife, there is a notable gap in understanding the implications of pruning practices in traditional olive groves. This study presents a unique observation, the first documented adverse effect of pruning on an olive tree within a traditional grove in Lesvos, Greece, inhabited by Persian squirrels (Sciurus anomalus). Beyond immediate impacts, we conducted comprehensive measurements of both the pruned olive tree and the squirrel’s nest (den), along with an assessment of the entire grove. The findings highlight the need for ecologically informed olive grove management to sustain biodiversity in these historically significant environments.