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Purpose: It is anticipated that global climate change will increase the frequency of wildfires in native forests of eastern Australia. Understorey legumes such as Acacia species play an important role in maintaining ecosystem nitrogen (N) balance through biological N fixation (BNF). This is particularly important in Australian native forests with soils of low nutrient status and frequent disturbance of the nutrient cycles by fires. This study aimed to examine N enrichment and N natural abundance techniques in terms of their utilisation for evaluation of N fixation of understorey acacias and determine the relationship between species ecophysiological traits and N fixation. Materials and methods: A trial was established at sites 1 and 2 located at Toohey Forest, Queensland, Australia, a eucalypt-dominated native forest, to examine the determination of BNF using N enrichment and N natural abundance methods. Toohey Forest is an urban forest and subjected to frequent fuel reduction burns to protect the adjacent properties. Plant physiological status was measured to determine the relationship between physiological and N fixation activities. Results and discussion: Both N enrichment and N natural abundance techniques may be used to estimate N fixation of acacia tree species. The estimation of BNF using N enrichment was higher than those of the N natural abundance method. A grass reference plant, Themeda triandra, as well as tree reference plants provided an appropriate δN signal. Potential B values for Acacia spp. between −0.3‰ and 1.0‰ provided an acceptable BNF estimation. This suburban forest is located nearby a busy highway leading to N deposition over time with consequent negative δN signal. This N deposition may explain the separation between the δN signal of the acacias and that of the reference plants which led to the successful use of the N natural abundance technique. Acacia leiocalyx demonstrated greater N fixation as well as photosynthesis and instantaneous water use efficiency than Acacia disparimma. However, no strong relationship between plant photosynthesis and N fixation was observed in this study. A high within-treatment variation may have masked the relationships between plant BNF activities and photosynthesis Conclusions: The N natural abundance technique is preferred to be used for future studies as it is simple and inexpensive compared with N enrichment method. The dependence of both species on BNF at site 2, where fuel reduction burning had not taken place for 8 years, suggests that the frequent burning impoverished the soil, and this has wider implications as higher fire frequencies are to be expected in other Australian ecosystems as a result of global climate change. [ABSTRACT FROM AUTHOR]