A rapidly changing world and constantly expanding knowledge requires education to no longer focus on teaching subject-matter knowledge but also to promote students’ critical thinking (CT) and an accurate understanding of the nature of science (NOS). However, several studies have shown that these skills are still poorly acquired during formal education. Given the cause–effect sequence from teacher education to teacher action to student learning, it seems reasonable to consider individual factors on the part of (pre-service) teachers as possible contributors to such skill gaps. In our study, we therefore investigated how pre-service biology teachers perform on tasks assessing their CT skills and NOS beliefs. In addition, we addressed the questions of whether test performance and/or the relationships between CT skills and NOS beliefs differ as a function of the number of learning opportunities. Our results show that our participants’ CT skills were only in the low–average range. Moreover, 86% of them did not have an informed understanding of NOS. Although participants in the master’s program demonstrated clearly superior CT skills than those in the bachelor’s program, no such difference was found in terms of NOS beliefs. However, there was a consistent advantage for pre-service teachers who were aspiring to a teaching qualification in two (as opposed to only one) scientific subjects. Our findings provide useful implications, particularly with respect to the influence of learning opportunities in university teacher education and the effectiveness of CT- and NOS-based instructional settings. On a more prospective note, our findings underscore that, given the grand global tasks of the 21st century, it seems more important than ever to ensure that pre-service science teachers have sufficient expertise in CT and NOS in order to increase the likelihood that these teachers will be able to successfully help their future students develop these skills.