Despite the central place held by bioinformatics in modern life sciences and related areas, it has only recently been integrated to a limited extent into high-school teaching and learning programs. Here we describe the assessment of a learning environment entitled Bioinformatics in the Service of Biotechnology. Students learning outcomes and attitudes toward the bioinformatics learning environment were measured by analyzing their answers to questions embedded within the activities, questionnaires, interviews and observations. Students difficulties and knowledge acquisition were characterized based on four categories: the required domain-specific knowledge (declarative, procedural, strategic or situational), the scientific field that each question stems from (biology, bioinformatics or their combination), the associated cognitive-process dimension (remember, understand, apply, analyze, evaluate, create) and the type of question (open-ended or multiple choice). Analysis of students cognitive outcomes revealed learning gains in bioinformatics and related scientific fields, as well as appropriation of the bioinformatics approach as part of the students scientific toolbox. For students, questions stemming from the old world biology field and requiring declarative or strategic knowledge were harder to deal with. This stands in contrast to their teachers prediction. Analysis of students affective outcomes revealed positive attitudes toward bioinformatics and the learning environment, as well as their perception of the teachers role. Insights from this analysis yielded implications and recommendations for curriculum design, classroom enactment, teacher education and research. For example, we recommend teaching bioinformatics in an integrative and comprehensive manner, through an inquiry process, and linking it to the wider science curriculum.