This study explores the influence of science and plant interest, alongside emotional responses, on the learning experiences of prospective primary education teachers during a botanical inquiry trail. Conducted at the University of Extremadura (Spain), the research involved 137 participants and valued their science and plant interest, knowledge acquisition, and emotional reactions before and after the trail. Results indicate that participants with higher interest in science and plants demonstrated greater knowledge gains and a more positive evaluation of the activity. Emotional responses, particularly reduced nervousness and increased enjoyment, further contributed to their engagement with botanical learning. The findings highlight the importance of encouraging interest in science and plants in teacher training programs to enhance environmental literacy and promote effective science education. This research underscores the importance of experiential learning strategies like botanical inquiry trails in motivating future educators to integrate active, inquiry-based approaches in their teaching practices.