Computer-aided multimedia learning provides learners with opportunities to individualize their own learning according to their own learning pace and needs, while at the same time it may cause cognitive overload for low achievers. Within the context of a computer-aided multimedia Biology lesson, the current study examined the effects of guided and unguided instruction on cognitive achievement and intrinsic motivation. Gender was taken into account and prior knowledge as a potential indicator of intrinsic load. The results showed that guided instruction had an effect on students reporting less tension and better perceived competence in successfully managing the learning task, as well as maintaining boys’ interest in the learning task even though no better learning occurred. Decrease rates of newly acquired knowledge were significantly lower with an unguided instruction compared to a guided learning, irrespective of students’ level of prior knowledge or gender.