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Biomass–fungi biocomposite materials are derived from sustainable sources and can biodegrade at the end of their service. They can be used to manufacture products that are traditionally made from petroleum-based plastics. There are potential applications for these products in the packaging, furniture, and construction industries. In the biomass–fungi biocomposite materials, the biomass particles (made from agricultural waste such as hemp hurd) act as the substrate, and a network of fungal hyphae grow through and bind the biomass particles together. Typically, molding-based methods are used to manufacture products using these biocomposite materials. Recently, the authors reported a novel extrusion-based 3D printing method using these biocomposite materials. This paper reports a follow-up investigation into the effects of mixing parameters (mixing time and mixing mode) on fungal growth in biomass–fungi mixtures prepared for 3D printing and the effects of printing parameters (printing speed and extrusion pressure) on fungal growth in printed samples. The fungal growth was quantified using the number of fungal colonies that grew from samples. The results show that, when mixing time increased from 15 to 120 s, there was a 52% increase in fungal growth. Changing from continuous to intermittent mixing mode resulted in an 11% increase in fungal growth. Compared to mixtures that were not subjected to printing, samples printed with a high printing speed and high extrusion pressure had a 14.6% reduction in fungal growth, while those with a low printing speed and low extrusion pressure resulted in a 16.5% reduction in fungal growth. [ABSTRACT FROM AUTHOR]