Nestedness is a statistical measure used to interpret bipartite interaction data in several ecological and evolutionary contexts, e.g. biogeography (species-site relationships) and species interactions (plant-pollinator and host-parasite networks). Multiple methods have been used to evaluate nestedness, which differ in how the metrics for nestedness are determined. Furthermore, several different null models have been used to calculate statistical significance of nestedness scores. The profusion of measures and null models, many of which give conflicting results, is problematic for comparison of nestedness across different studies. We developed the FALCON software package to allow easy and efficient comparison of nestedness scores and statistical significances for a given input network, using a selection of the more popular measures and null models from the current literature. FALCON currently includes six measures and five null models for nestedness in binary networks, and two measures and four null models for nestedness in weighted networks. The FALCON software is designed to be efficient and easy to use. FALCON code is offered in three languages (R, MATLAB, Octave) and is designed to be modular and extensible, enabling users to easily expand its functionality by adding further measures and null models. FALCON provides a robust methodology for comparing the strength and significance of nestedness in a given bipartite network using multiple measures and null models. It includes an “adaptive ensemble” method to reduce undersampling of the null distribution when calculating statistical significance. It can work with binary or weighted input networks. FALCON is a response to the proliferation of different nestedness measures and associated null models in the literature. It allows easy and efficient calculation of nestedness scores and statistical significances using different methods, enabling comparison of results from different studies and thereby supporting theoretical study of the causes and implications of nestedness in different biological contexts.