Microbial biofilm communities are composed of fungi, bacteria, and phytoplankton taxonomic groups (e.g., chlorophytes, diatoms and cyanobacteria), which inhabit the surface of intertidal mudflats. Such biofilms have critical roles in shorebird diets, mudflat stabilization, primary productivity, and carbon storage. This raster dataset represents the likely presence and absence of biofilm located on the mudflats of South San Francisco Bay in Spring 2021, during peak shorebird migration. To produce this dataset, we used a multiscalar remote sensing approach, which coupled in-situ data with hyperspectral data from an ASD field spectrometer, a HySpex VNIR/SWIR imaging spectrometer (5 mm), and the AVIRIS-Next Generation (NG) airborne imaging spectrometer (3.7 m). We calculated the B-index (Biofilm presence or absence index) based on the depth of the chlorophyll-a absorption feature at 680nm. We calibrated the index with HySpex spectra paired with in-situ measurements of biofilm chlorophyll-a concentrations (ng/g), a proxy for biofilm biomass. After validating the index with HySpex images, we defined a threshold index value to represent biofilm presence. Threshold B-index values of 0.015, 0.02 and 0.025 were set to represent sparse to abundant biofilm presence. We applied the B-index to the AVIRIS-NG image collected on April 15, 2021 over South San Francisco Bay. Maps created using hyperspectral remote sensing data will support managers’ need to visualize shorebird habitat quality and research on drivers of biofilm quality and quantity.