Background This study aimed to evaluate the effect of double application of universal adhesives on bond strength to dentine after ionizing radiation. Methods One hundred and forty caries-free human third molars were used. Half of the teeth were irradiated (IR) (70.2 Gy). The IR and non-IR teeth were assigned into seven groups (n = 10). Universal adhesives Single Bond Universal (3M Oral Care), Gluma Bond Universal (Heraeus Kulzer) and Prime&Bond Elect (Dentsply) were employed (following manufacturer's instructions and double application). Two-step self-etch adhesive Clearfil SE Bond (Kuraray Noritake) was used as control. After composite build-ups were bonded to mid-coronal occlusal dentine surfaces, the specimens were stored in water (37 degrees C/24 h) and sectioned into microspecimens. The specimens were subjected to mu TBS testing (1.0 mm/min). Failure analysis was performed using stereomicroscope and scanning electron microscopy. Data were analysed with two-way ANOVA and Tukey's test (P = 0.05). Results The radiotherapy caused reduction in the bond strength of universal adhesives when the adhesives were used with single application after radiotherapy (P < 0.05). The double application resulted in increased bond strength of the universal adhesives to IR dentine (P < 0.05). Conclusions The double application was beneficial in improving the dentine bond strength of universal adhesives affected by radiotherapy after ionizing radiation.