Microbiological and biomolecular approaches to cultural heritage research have expanded the established research horizon, from the prevalent focus on the conservation of the cultural objects and the protection of human health, to the relatively recent applications to provenance inquiry and assessment of environmental impacts on heritage objects in a global context of a changing climate. Standard microbiology and molecular biology methods were developed for other materials, specimens, disciplines and contexts. Although they could in principle be applied to cultural heritage research, certain characteristics common to several heritage objects, such as uniqueness, fragility, high value and restricted access, demand tailored approaches. In addition, samples from heritage objects often yield low microbial biomass, rendering them highly susceptible to cross-contamination. Therefore, dedicated methodology that addresses these material limitations and operational hurdles along all procedural steps are needed. Here were propose a step-by-step standardized laboratory and bioinformatic workflow to analyse the microbiome of cultural heritage objects. The methodology was developed targeting the challenging side of the spectrum of cultural heritage objects, such as the delicate written record, while retaining modularity and flexibility to adapt and/or upscale the proposed workflow to heritage artefacts of a more robust nature or larger dimensions. We hope this review and workflow will facilitate the interdisciplinary inquiry and interactions among the cultural heritage research community.