This longitudinal study aimed to investigate variables associated with colonisation of mutans streptococci (MS) compared with lactobacilli (LB) colonisation in a cohort of children (n = 214) from the time of first tooth eruption at approximately 6 months until 24 months of age. Repeated plaque and salivary samples were collected from the same infants at 6, 12, 18 and 24 months and assayed for MS and LB using a microbiological culture kit. Children having both MS and LB increased from 4% at 6 months to 13% at 12 and 18 months to 20% at 24 months (p = 0.004). LB presence at 6 months was correlated with MS presence at 12, 18 and 24 months (r = 0.21 to r = 0.46, p = 0.02), while MS presence at 6 months correlated with LB presence at all other times (r = 0.19 to r = 0.31, p = 0.03). At 6 and 12 months, the key variables for MS colonisation included unrestored dental cavities in the mother (p = 0.03), mother not persisting with toothbrushing (p = 0.001) and bottle taken to bed at night (p = 0.033), while the only significant variable for LB colonisation was natural birth (p = 0.01). At 24 months, the significant variables for MS colonisation were condiments added to pacifier (p = 0.022) and child being uncooperative for toothbrushing (p = 0.025), while the significant variables for LB colonisation were pregnancy problems (p = 0.028) and child being uncooperative for toothbrushing (p = 0.013). The ages 6–12 months thus represent a time period when key variables may be controlled to reduce MS and LB colonisation.