PyBioNetFit (PyBNF) is a general-purpose program for parameterizing biological models specified using the BioNetGen rule-based modeling language (BNGL) or the Systems Biology Markup Language (SBML). PyBioNetFit offers a suite of parallelized metaheuristic algorithms (differential evolution, particle swarm optimization, scatter search) for parameter optimization. In addition to model parameterization, PyBNF supports uncertainty quantification by bootstrapping or Bayesian approaches, and model checking. PyBNF includes the Biological Property Specification Language (BPSL) for defining qualitative data for use in parameterization or checking. It runs on most Linux and macOS workstations as well on computing clusters.