Biochar is charcoal made from waste biomass and intended to be added to soil to improve soil function and reduce emissions from the biomass caused by natural degradation to CO2. Nitrogen (N) forms in biochar can be complex and their lability likely to be influenced by pyrolysis temperature which, together with the nature of carbon (C), will influence N mineralisation or immobilisation. These complex relationships are poorly understood, yet impact strongly on the potential agronomic value of biochar. In this study, N in different biochar samples produced from human and animal waste streams (biosolids and cow manure; each mixed with eucalyptus wood chips in a 1:1 dry wt. ratio) at different pyrolysis conditions (highest heating temperature 250, 350, 450 and 550 °C) was extracted with 6 M HCl. The acid hydrolysable, extractable N (THN) was fractionated into ammonia N (AN), amino acid N (AAN), amino sugar N (ASN) and uncharacterisable hydrolysable N (UHN). Biochar samples were also treated with 0.167 M K2Cr2O7 acid to determine N potentially available in the long term. An incubation study of the different biochar samples mixed with acid washed sand was conducted at 32 °C for 81 days to study both C and N turnover. During incubation, the CO2 released was trapped in NaOH and quantified. Hydrolysable N decreased as pyrolysis temperature increased from 250 to 550 °C. Fractionation into AN, AAN, ASN and UHN revealed progressive structural rearrangement of N with pyrolysis temperature. Based on HCl hydrolysis and dichromate oxidation results, C and N in biochar became more stable as pyrolysis temperature increased. The ratio of volatile C to THN was a useful indicator of whether net N mineralisation or immobilisation of N in biochar occurred. THN thus seems a sound estimate of the labile N fraction in biochar in the short term; however, dichromate-oxidisable N is probably more meaningful in the long run. Further studies using different types of biochar need to be conducted under more realistic conditions to obtain more information on N availability in biochar once in soil.