Biochars derived from animal manures may accumulate potentially toxic metals and cause a potential risk to ecosystem. The synchrotron-based X-ray spectroscopy, sequential fractionation schemes, bioaccessibility extraction and leaching procedure were performed on poultry and swine manure-derived biochars (denoted PB and SB, respectively) to evaluate the variance of speciation and activity of Cu and Zn as affected by the feedstock and pyrolysis temperature. The results showed that Cu speciation was dependent on the feedstock with Cu-citrate-like in swine manure and species resembling Cu-glutathione and CuO in poultry manure. Pyrolyzed products, however, had similar Cu speciation mainly with species resembling Cu-citrate, CuO and CuS/Cu S. Organic bound Zn and Zn (PO ) -like species were dominant in both feedstock and biochars. Both Cu and Zn leaching with synthetic precipitation leaching procedure (SPLP) and toxicity characteristic leaching procedure (TCLP) decreased greatly with the rise of pyrolysis temperature, which were consistent with the sequential extraction results that pyrolysis converted Cu and Zn into less labile phases such as organic/sulfide and residual fractions. The potential bioaccessibility of Zn decreased for both the PB and SB, closely depending on the content of non-residual Zn. The bioaccessibility of Cu, however, increased for the SB prepared at 300°C–700°C, probably due to the increased proportion of CuO. Concerning the results of sequential fractionation schemes, bioaccessibility extraction and leaching procedure, pyrolysis at 500°C was suggested as means of reducing Cu/Zn lability and poultry manure was more suitable for pyrolysis treatment.