The attenuation of photosynthetically active radiation (PAR) through the filamentous alga Cladophora glomerata was investigated both in situ and in an experimental flow tank under natural and artificial lighting using a fiber-optic sensor. Over 35 separate experiments were completed to characterize downward light attenuation and water velocity effects. Measured extinction coefficients ranged from 4.3 to 150.7 m−1 for biomass samples ranging from 40 to 253 mg chlorophyll-a m−2 (41 to 381 g ash free dry weight m−2) at shear velocities from 0 to 51.8 cm s−1. An exponential relationship between light attenuation coefficient (Kalg) and algal biomass thickness (δ) was found (lnKalg = −1.1831 lnδ + 8.3789; r2 = 0.41, p < 0.001). Unexpectedly, chlorophyll-a (Chla) and ash free dry weight (AFDW) areal density were poor predictors of Kalg. No significant correlations (p > 0.01) were found amongst any of the other experimental variables (e.g., shear velocity, Reynolds number, etc.). Cladophora mat thickness (δ) and volumetric biomass (Chla m−3) provided the greatest explanation of downward light attenuation in flowing water, reducing irradiance in proportion to how closely the filaments are pressed together. Observed δ generally decreased with increasing shear velocity, albeit not predictably. Results have important implications on aquatic systems where macroalgae generate variable light regimes on understory aquatic species.