Soil organic matter (SOM) is an important store of carbon and is vital to maintaining soil health. Growing crops generally causes a reduction in SOM. However, organic farming systems often adopt practices that partially mitigate this loss. Biodegradable plastic film mulch (PFM) can increase yields by improving soil hydrothermal conditions, increasing nitrogen use efficiency and suppressing weeds. It can also speed up SOM breakdown and induce changes to the soil microbiome. Further, the increased return of C from rhizodeposition and crop residues from PFM-grown crops can compensate for SOM breakdown, although outcomes vary substantially with agronomic and environmental conditions. To address these uncertainties, a plot-scale field experiment was conducted on an organic farm with a 3-year vegetable rotation measuring SOM content from treatments with and without biodegradable PFM, inputs of poultry manure or green waste compost, and with or without an overwinter green manure. Biodegradable PFM caused a significant increase in yield in all the crops grown (43%–46%) and the overwinter green manures (18%), resulting in more organic matter incorporated into the soil. Despite this, there was no significant difference in the SOM content between the biodegradable PFM- and non-PFM-mulched plots over the 3 years, nor was there any significant change in soil bacterial diversity. In contrast, the large difference in the mass of green waste compost and poultry manure addition resulted in a 15% increase in SOM after 3 years. Biodegradable PFM did not affect alpha (Shannon) or beta diversity of soil bacterial community.