Background This study examined the effect of operator experience, dominance, tooth position and access, on frequency and extent of iatrogenic damage to approximal tooth surfaces during conventional Class II cavity preparations, and the effectiveness of protective devices in minimising damage. Methods 10 students and 10 experienced dentists each prepared 24 Class II cavity preparations in typodont teeth without protection, 10 utilising stainless steel matrix bands and 10 utilising protective wedges. The frequency and extent of damage were analysed with respect to the above variables. Subsequently, 20 natural and 20 typodont teeth were utilised to establish the relationship in depth of damage caused by a high-speed diamond bur on typodont versus natural teeth. Results Dentists caused iatrogenic damage on 74% of approximal surfaces without protection, which fell to 50% and 46% respectively when matrix bands and wedges were used as guards. The corresponding rates of damage for students were 94%, 80% and 44%. There was no difference in depth of damage caused on the two types of teeth when bur was in contact with teeth for a very short time. Conclusions Greater operator experience and the use of guards reduces iatrogenic damage to proximal surfaces during preparation with high-speed rotary instruments.