Polyhydroxybutrate (PHB) is a biodegradable polymer that has huge potential as an alternative to replace petroleum-based plastics. This study presented mechanical strength improved PHB composites reinforced by nanocellulose fibrils, which is another biodegradable engineering material. The chemical crosslinking between PHB polymer chains and nanocellulose fibrils was conducted, and the PHB/CNF composite film was fabricated by solvent casting method. After the crosslinking using toluene-2,4-diisocyanate (TDI), the interaction of the two different moieties was significantly improved. SEM, FT-IR, DSC and XRD were used in the characterizations of TDI crosslinked PHB/CNF composites, demonstrating that both the crystallinity and thermal stability of PHB were increased in the composites. Amplicon sequencing targeting bacterial 16S rRNA and fungal ITS suggested that PHB crosslinked with CNF, or/and TDI impacted the growth of different microbial species.