Diurnal changes in the biochemical properties and the benthic macrofaunal assemblage of sediments in urbanised mangrove forests and their adjacent mudflats in Sydney Harbour were investigated. Behavioural and physiological changes in the microphytobenthos between day and night were predicted to cause diurnal changes in the micro-scale depth distribution of chlorophylls a and b and colloidal carbohydrate. In addition, because macrofauna can alter sediment properties, diurnal changes in the macrofaunal assemblages were investigated. The microphytobenthos at the study sites were predominantly filamentous green algae, although diatoms were present. Samples for biochemical analysis were collected from the top 2 mm of sediment using mini-cryolanders, during low tide in the day and at night. Three biochemical properties of the sediments were measured spectrophotometrically: chlorophylls a and b (surrogate for microphytobenthos biomass) and colloidal carbohydrate. The amount of chlorophylls tended to be less at night than during the day, but site to site variability was large and these differences were generally small and not significant. Depth profiles indicated that there was some redistribution of pigments in the surface 2 mm between day and night, possibly due to migration of microphytobenthos or physiological changes. There was no significant difference in chlorophylls between the mangrove forest and adjacent mudflat, with the exception of chlorophyll b at one sampling time, which was larger in the mangrove forest than on the mudflat. Colloidal carbohydrate was significantly larger in the mangrove forest and significantly less on the mudflat during the day at one site at one time, but otherwise showed no significant differences between day and night or between the mangrove forest and mudflat. Whilst there were some differences in the benthic macrofaunal assemblages between day and night, these differences were only significant for spionids and polychaetes at one time. There were, however, significant differences in assemblages of benthic macrofauna between the mangrove forest and mudflat, probably due to structural differences between these habitats such as the presence of pneumatophores, shade and leaf litter. In summary, there was some minor diurnal variation in the measured biochemical properties of the sediment, but not in the macrofaunal assemblage. Diurnal changes should, therefore, be considered when investigating biochemical properties in these habitats, but they are not a major influence. These findings contrast to previous studies on diatom dominated mudflats in Europe, where stronger diurnal changes in biochemical properties were found. Diurnal changes in the macrofauna assemblages were largely insignificant and therefore could not explain the changes in the biochemical properties. Diurnal effects on the macrofauna in these habitats are more likely to be via altered behaviours and this requires further investigation.