Background:Cycloplegia has been shown to have no effect on axial length measurement made with the IOLMaster in adults. The current study aimed at evaluating the effect of cycloplegia on axial length and anterior chamber depth (ACD) measurements made with the IOLMaster and an ultrasonic biometer in children. Methods: Pre-and post-cycloplegic axial length and ACD were measured with the IOLMaster followed by the Sonomed A-5500 in 31 children aged from seven to 15 years by the same examiner. The 95% limits of agreement (LoA) were determined, if there were no significant correlations found between the mean differences and their means. Results: Seven subjects were excluded. Results from the remaining 24 subjects show that the effects of cycloplegia, instruments, and interaction between cycloplegia and instrument on axial length measurement were insignificant (repeated measure ANOVA F 1,23 < 2.19, p > 0.15). The 95% LoA in cycloplegia were better with the IOLMaster (-0.04 to 0.04 mm) than with the Sonomed A-5500 (-0.13 to 0.14 mm). The 95% LoA between the two instruments were similar with and without cycloplegia (pre-cycloplegia: -0.20 to 0.27 mm; post-cycloplegia: -0.17 to 0.22 mm).There was no significant interaction between cycloplegia and instrument in ACD measurement (repeated measure ANOVA F1,23 = 0.85, p = 0.37), however, ACD was 0.05 to 0.06 mm shorter before cycloplegia (repeated measure ANOVA F1,23 = 44.70, p < 0.001) and was 0.06 to 0.08 shorter measured with the IOLMaster (repeated measure ANOVA F1,23 = 28.81, p < 0.001). Conclusion: Effects of cycloplegia on axial length measurement in children made with IOLMaster and Sonomed A-5500 were insignificant. In contrast, ACD measurement was significantly affected by cycloplegia and different instruments.