Global biodiversity declines at unprecedented rates, mainly due to human-induced environmental change. Biodiversity conservation is, thus, highly dependent on responsible and sustainable citizenship. Educational efforts are regarded as an important means to foster awareness and pro-environmental behavior. The present study monitors two factors considered to be particularly relevant for promoting sustainable behavior: cognitive knowledge and environmental values. 205 students (Mage = 15.3) participated in a biodiversity education module including a citizen science (CS) activity on DNA barcoding. With a pre-post-retention design, we measured cognitive achievement and environmental values, which are expressed by environmental utilization (UTL) and preservation (PRE) as well as the appreciation for nature (APR). Overall, we found positive relations between knowledge and PRE as well as APR, whereas UTL was negatively related to knowledge. In the whole module and the sub-modules, cognitive achievement followed the usual pattern, with a substantial short-term knowledge increase from pre-test (T0) to post-test (T1) following a moderate decrease in the retention test (T2). Unexpectedly, a considerable sub-sample (n = 103) deviated from the assumed knowledge drop at T2 and showed an additional knowledge gain in a sub-module directly focusing on the CS activity. Students in this sub-sample revealed significantly higher PRE and APR scores compared to the rest of the students. We discuss these findings in relation to the implications for educational CS.