We aimed to assess the association between the roll-out of the national nursery toothbrushing program and a reduction in dental decay in five-year-old children in a Scotland-wide population study. The intervention was supervised toothbrushing in nurseries and distribution of fluoride toothpaste and toothbrushes for home use, measured as the percentage of nurseries participating in each health service administrative board area. The endpoint was mean d(3)mft in 99,071 five-year-old children, covering 7% to 25% of the relevant population (in various years), who participated in multiple cross-sectional dental epidemiology surveys in 1987 to 2009. The slope of the uptake in toothbrushing was correlated with the slope in the reduction of d(3)mft. The mean d(3)mft in Years -2 to 0 (relative to that in start-up Year 0) was 3.06, reducing to 2.07 in Years 10 to 12 (difference = -0.99; 95% CI -1.08, -0.90; p < 0.001). The uptake of toothbrushing correlated with the decline in d(3)mft (correlation = -0.64; -0.86, -0.16; p = 0.011). The result improved when one outlying Health Board was excluded (correlation = -0.90; -0.97, -0.70; p < 0.0001). An improvement in the dental health of five-year-olds was detected and is associated with the uptake of nursery toothbrushing.